Preparation and characterization of inclusion complexes of naringenin with β-cyclodextrin or its derivative

Article abstract of DOI:10.1016/j.carbpol.2013.07.010

The inclusion complexation behavior, characterization and binding ability of naringenin with β- cyclodextrin and its derivatives were investigated in both solution and the solid state by means of XRD, DSC, SEM, ¹H and 2D NMR and UV-vis spectros

Full text of DOI:10.1016/j.carbpol.2013.07.010

Carbohydrate Polymers 98 (2013) 861–869
Contents lists available at SciVerse ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Preparation and characterization of inclusion complexes of
naringenin with ␤-cyclodextrin or its derivative
Li-Juan Yanga,∗, Shui-Xian Maa, Shu-Ya Zhoua, Wen Chenb, Ming-Wei Yuana,
Yan-Qing Yina, Xiao-Dong Yangb,∗
a Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities,
Kunming 650500, 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:
Received 13 June 2013
Received in revised form 30 June 2013
Accepted 3 July 2013
Available online 12 July 2013
The inclusion complexation behavior, characterization and binding ability of naringenin with ␤-
cyclodextrin and its derivatives were investigated in both solution and the solid state by means of XRD,
DSC, SEM, 1H and 2D NMR and UV–vis spectroscopy. The results showed that the water solubility and
thermal stability of naringenin were obviously increased in the inclusion complex with cyclodextrins. This
satisfactory water solubility and high thermal stability of the naringenin/CD complexes will be potentially
useful for their application as herbal medicines or healthcare products.
Keywords:
Naringenin
© 2013 Elsevier Ltd. All rights reserved.
Cyclodextrin
Inclusion complex
Characterization
Inclusion mode
1. Introduction
recent years, in vitro and in vivo studies have shown that naringenin
has an insulin-like effect to decrease apoliprotein B (ApoB) secre-
The flavonoid naringenin (5,7,4ꢀ-trihydroxyflavanone or 5,7-
dihydroxy-2-(4-hydroxyphenyl)chroman-4-one, Fig. 1), a tradi-
tional Chinese medicine, is one of the most abundant flavonoids
in grapefruits and citrus fruits (El-Mahdy et al., 2008). Daily inges-
tion of citrus flavonoids has been estimated to be approximately
68 g on an average in the USA. Naringenin is mainly ingested
from fruit juices. The concentration of naringenin in the grape-
fruit juice is found to be 1283 mM (349 mg/L) (Jayachitra & Nalini,
2012). Naringenin plays a key role as an estrogenic substance in
humans and as an endogenous regulator in plants, and is known
as a safe natural product with various bioactive effects (Fang,
Tang, Gao, & Xu, 2010; Kretzschmar et al., 2007). It has been
reported to have anticancer, antimutagenic, anti-inflammatory,
antiatherogenic, antifibrogenic and free radical scavenging prop-
erties (Jayachitra & Nalini, 2012). Naringenin is reported to induce
cytotoxicity and apoptosis in various cancer cell line and its treat-
ment at a similar dose showed no toxic effect on normal cells (Jin
et al., 2009; Park et al., 2008; Sabarinathan & Vanisree, 2011). In
tion in hepatocytes and decreased blood glucose levels in healthy
male Wistar rats (Zygmunt, Faubert, MacNeil, & Tsiani, 2010). Fur-
thermore, naringenin is able to traverse the blood–brain barrier
and exert a diverse array of neuronal effects through their abil-
ity to interact with the Protein Kinase C (PKC) signaling pathways
(Sabarinathan & Vanisree, 2011). Additionally, naringenin was
shown to reduce the accumulation of collagen fibers by reduced
hepatic stellate cell activity in dimethylnitrosamine-induced liver
injury rates and to exhibit antifibrogenic effects (Liu et al.,
2006).
However, the use of naringenin as a natural herbal medicine
is greatly limited by its low water solubility and bioavailability
(Hsiu, Huang, Hou, Chin, & Chao, 2002; Yen, Wu, Lin, Cham, &
Lin, 2009). Although much effort has been made to improve its
water solubility and stability by introducing some nanodispersion
or enzymatic condensation techniques (Vila Real, Alfaia, Calado, &
Ribeiro, 2007; Yen et al., 2009), it is still not possible to sufficiently
dissolve naringenin in water, which prevents its usage for some
therapeutic applications. Therefore, the search for an efficient and
nontoxic carrier for naringenin has become important in order to
further its clinical applications.
∗
Corresponding authors. Tel.: +86 871 6503 1119; fax: +86 871 6503 5538.
E-mail addresses: ljyyang@gmail.com (L.-J. Yang), xdyang120@gmail.com
It is well known that cyclodextrins (CDs) are truncated-
cone polysaccharides mainly composed of six to eight d-glucose
(X.-D. Yang).
0144-8617/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carbpol.2013.07.010

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L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
OR1
3'
O
OH
2'
H-5
H-5
1
4'
5'
C
8
R3O
9
2
HO
O
OR2
1'
7
6
6'
A
O
7
10 B
H-3
H-3
3
4
5
β-CD R1=R2=R3=H
OH
O
DMβCD R1=R2=Me, R3=H
TMβCD R1=R2=R3=Me
naringenin
Fig. 1. The structures of naringenin, ␤-CD and its derivatives.
monomerslinked by ␣-1,4-glucosebonds. They have a hydrophobic
central cavity and a hydrophilic outer surface and can encap-
sulate various inorganic/organic molecules to form host–guest
complexes or supramolecular species. This usually enhances drug
solubility in aqueous solutions and affects the chemical charac-
teristics of the encapsulated drug in the 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), photo-
chemical sensors (Ueno, 1996), etc. Recently, some studies of
inclusion complex formation between CDs and flavonoids have
been reported (Cannavà et al., 2010; Koontz, Marcy, O’Keefe,
& Duncan, 2009; Mourtzinos et al., 2008; Tommasini et al.,
2004).
2.2. Methods
2.2.1. Preparation of naringenin/ˇ-CD, naringenin/DMˇCD and
naringenin/TMˇCD complexes
Naringenin (0.03 mM, 8.2 mg) and CD (0.01 mM) were com-
pletely dissolved in a mixed solution of ethanol and water (ca.
10 mL, v:v = 1:4, given the poor water solubility of naringenin,
ethanol was used), and the mixture was stirred for 5 days at room
temperature. After evaporating the ethanol from the reaction mix-
ture, the uncomplexed naringenin was removed by filtration. The
filtrate was evaporated under reduced pressure to remove the sol-
vent and dried in vacuum to produce the naringenin/CD complexes.
2.2.2. Spectral titration
Absorption spectra measurements were carried out with a
Shimadzu UV 2401 (Japan) using
a conventional 1 cm path
More recently, we reported that the inclusion complexation
of CDs with natural medicines such as taxifolin (Yang et al.,
2011), crassicauline A (Chen et al., 2011), nimbin (Yang et al.,
2010), alpinetin (Ma et al., 2012), and artemether (Yang, Lin,
Chen, & Liu, 2009) significantly enhanced the water solubility
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
et al., 2009; Yang et al., 2010). As a continuation of our studies
on natural medicines/cyclodextrin inclusion complex, an inclusion
complex of naringenin with ␤-CD and its derivatives was investi-
gated.
(1 cm × 1 cm × 4 cm) quartz cell in a thermostated compartment,
which was kept at 25 ◦C by a Shimadzu TB-85 Thermo Bath unit.
Given the poor water solubility of naringenin, a water/ethanol
(v:v = 4:1) solution was used in the spectral measurements. The
concentration of naringenin was held constant at 0.03 mM. Then, an
appropriate amount of CD was added, and the final concentrations
varied from 0 to 2.80–4.00 mM (␤-CD: 0, 0.23, 0.47, 0.67, 0.96, 1.37,
1.96, 2.80 mM; DM␤CD: 0, 0.23, 0.47, 0.96, 1.37, 2.80, 4.00 mM;
TM␤CD: 0, 0.33, 0.47, 0.67, 0.96, 1.37, 1.96, 2.80 mM). The absorp-
tion spectra measurements were taken after 1 h. The measurements
were done in the 220–400 nm spectral range. All experiments were
carried out in triplicate.
In this paper, we aim to report the preparation and charac-
terization of some water-soluble inclusion complexes formed by
naringenin and ␤-cyclodextrin and its derivatives: heptakis-(2,6-
di-O-methyl)-␤-CD (DM␤CD) and heptakis(2,3,6-tri-O-methyl)-␤-
CD (TM␤CD) (Fig. 1). We were particularly interested in exploring
the solubilization effect of CDs on naringenin and the binding abil-
ity of the resulting inclusion complexes, which would provide a
useful approach for obtaining novel naringenin-based healthcare
products with high water solubility, high bioavailability and low
toxicity.
2.2.3. 1H and 2D NMR
All NMR experiments were carried out in D2O. Tetramethylsi-
lane was used as a reference. The 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 reso-
lution of 0.18 Hz/point. The residual HDO line had a line width at a
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, with 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 2 K in both dimensions using sine (F2) and
qsine (F1) window functions, respectively.
2. Materials and methods
2.1. Materials
2.2.4. Powder X-ray diffraction (XRD)
Naringenin (FW = 272, PC > 98%) was obtained from the National
Institute for Control of Pharmaceutical and Bioproducts (Beijing, PR
China); ␤-CD (average substitution degree = 1135), heptakis(2,6-
di-O-methyl)-␤-CD (DM␤CD, average substitution degree = 1331),
and heptakis(2,3,6-tri-O-methyl)-␤-CD (TM␤CD, average substitu-
tion degree = 1429) were purchased from ABCR GmbH & Co. KG and
used without further purification. Other reagents and chemicals
were of analytical reagent grade. All experiments were carried out
using ultrapure water.
The XRD patterns were obtained using a D/Max-3B diffractome-
ter 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◦.
2.2.5. Thermal analyses
Differential scanning calorimetry (DSC) and thermogravimetric
(TG) measurements were performed with a 2960 SDT V3.0F

L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
863
instrument and NETZSCH STA 449F3, respectively, at a heating
rate of 10 ◦C/min from room temperature to 400 ◦C in a dynamic
nitrogen atmosphere (flow rate = 70 mL/min).
naringenin gradually increased with the stepwise addition of ␤-
CD, DM␤CD and TM␤CD. The pH of the solution did not change
appreciably during any of the experimental procedures. 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 a 1:1 stoichiometry for the naringenin/CD
inclusion complex, the inclusion complexation of naringenin with
␤-CD could be expressed by Eq. (1), and the stability constant (Ks)
could be calculated from Eq. (2), where [naringenin · CD], [narin-
genin], [CD], [naringenin]0 and [CD]0 refer to the equilibrium
concentration of the naringenin/CD inclusion complex, the equilib-
rium concentration of naringenin, the equilibrium concentration
of CD, the original concentration of naringenin, and the original
concentration of CD, respectively, and ꢁA and ꢁε are the differ-
ential absorption and molar extinction coefficient of naringenin
in the absence and presence of CD. According to Lambert–Beer
Law, we can obtain that the concentration of the naringenin/CD
complex is equal to ꢁA/ꢁε ([naringenin · CD] = ꢁA/ꢁε). If the
naringenin and the CD form complex with a 1:1 stoichiometry,
the concentration of the naringenin/CD complex ([naringenin · CD])
is equal to the reduced concentration of the naringenin, and
it is also equal to the reduced concentration of CD. So the
equilibrium concentration of naringenin is equal to the original
concentration of naringenin minus the concentration of the narin-
genin/CD complex ([naringenin] = [naringenin]0 − [naringenin
· CD] = [naringenin]0 − ꢁA/ꢁε), and the equilibrium concentration
of CD is equal to the original concentration of CD minus the concen-
tration of the naringenin/CD complex ([CD] = [CD]0 − [naringenin
2.2.6. 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×.
2.2.7. Solubilization test
An excess amount of the 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 20 2 ◦C. The solution was then filtered on a
0.45 ␮m cellulose acetate membrane. The filtrate was evaporated
under reduced pressure to dryness and the residue was dosed by
the weighing method.
3. Results and discussion
3.1. Spectral titration
A quantitative investigation of the inclusion complexation
behavior of ␤-CD and its derivatives with naringenin was carried
out in a water/ethanol (v:v = 4:1) solution using a spectrophoto-
metric titration method owing to the rather low water solubility
of naringenin. As illustrated in Fig. 2, the absorbance intensity of
0.02
0.010
0.42
0.45
0.30
0.15
0.00
h
a
g
Esp
Calcd
Esp
Calcd
0.01
0.005
0.00
0.000
0.0
0.9
1.8
2.7
1
2
4
0[DMβ-CD3]/nm
0.21
0.00
a
[β-CD]/nm
1
2
250
300
/nm
350
400
220
275
330
385
λ
λ
/nm
0.0150
h
0.42
0.21
0.00
Esp
Calcd
0.0075
0.0000
0
1
2
3
[TMβCD]/nm
a
3
220
275
330
385
λ
/nm
Fig. 2. UV–vis spectral changes in naringenin (0.03 mM) upon addition of ␤-CD (1: 0–2.80 mM, from a to h), DM␤CD (2: 0–4.00 mM, from a to g) and TM␤CD (3: 0–2.80 mM,
from a to h) in a water/ethanol (v:v = 4:1, ca. pH 3.0) mixed solution, and the nonlinear least-squares analysis (inset) of the differential intensity (ꢁA at 289 nm) to calculate
the complex stability constant (Ks).

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L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
3.3. 1H and 2D NMR analysis
Table 1
The stability constant (Ks and log Ks) and Gibbs free energy change (−ꢁG◦) for the
inclusion complexation of CDs with naringenin guest in a water/alcohol (v/v = 4:1,
ca. pH 3.0) mixed solution.
In order to explore the possible inclusion mode of the narin-
genin/CD complex, we compared the 1H NMR spectra of naringenin
in the presence of the host CDs (Fig. 3). Owing to its poor water
solubility, naringenin is transparent to 1H NMR under most condi-
tions when D2O is used as a solvent. Assessment of the naringenin
complex by 1H NMR clearly demonstrated the presence of the
framework protons of the naringenin molecule, consistent with
the significant solubilization. As illustrated in Fig. 3, the majority
of naringenin protons displayed chemical shifts at ı 5.5–7.5 ppm,
which were distinct from the CD protons (usually at ı 3.0–5.0 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 naringenin/CD complexes, that is, 1:1 for the naringenin/␤-CD,
naringenin/DM␤CD and naringenin/TM␤CD complexes.
Host
Ks/M−1
log Ks
−ꢁG◦/kJ mol−1
␤-CD
DM␤CD
TM␤CD
2242
1409
936
3.35
3.15
2.97
19.12
17.97
16.96
· CD] = [CD]0 − ꢁA/ꢁε). Based on the formula of equilibrium con-
stant, we can obtain Ks = [naringenin · CD]/([naringenin] · [CD])
= (ꢁA/ꢁε)/(([naringenin]0 − ꢁA/ꢁε) · ([CD]0 − ꢁA/ꢁε)) (Eq. (2)).
We then derived Eq. (3) from Eq. (2). Finally, the Ks was obtained
from the analysis of the sequential changes of absorption (ꢁA) at
various CD concentrations, with a nonlinear least squares method
according to the curve-fitting equation (3).
To further explore the inclusion mode, the chemical shifts of
␤-CD protons in the absence and presence of naringenin were
examined (Fig. 3). Inclusion complexation with naringenin had
K
s
Naringenin + CDꢀNaringenin · CD
(1)
[Naringenin · CD]
ꢁA/ꢁε
Ks =
=
(2)
[Naringenin][CD]
([Naringenin]0 − (ꢁA/ꢁε))([CD]0 − (ꢁA/ꢁε))
ꢀ
ꢁε([Naringenin]0 + [CD]0 + 1/Ks)
ꢁε2([Naringenin]0 + [CD]0 + 1/Ks)2 − 4ꢁε2[Naringenin]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. 2 (inset) illustrates a typ-
ical curve-fitting plot for the titration of naringenin with ␤-CD,
DM␤CD and TM␤CD, which shows the excellent fit between the
experimental and calculated data and the 1:1 stoichiometry of
the naringenin/CD inclusion complexes. In the repeated measure-
ments, 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 CDs with naringenin are listed in Table 1.
a negligible effect on the ı values of the H-5 and H-6 protons
of ␤-CD (≤0.02 ppm). In contrast, the values of the H-1, H-2,
H-3 and H-4 protons exhibited relatively weak but significant
changes (0.03–0.04 ppm), which could have been caused by the
hydrogen bond between the hydroxyl arms of ␤-CD and the
oxygen atoms of naringenin. It is noteworthy that the H-3 pro-
tons shifted ca. 0.03 ppm, but that the H-5 protons shifted ca.
0.01 ppm after inclusion complexation. Because both the H-3 and
H-5 protons are located in the interior of the ␤-CD cavity, and
the H-3 protons are near the wide side of the cavity while the
H-5 protons are near the narrow side, this phenomenon may
indicate that naringenin should penetrate into the ␤-CD cavity
from the wide side. Similarly, all of the TM␤CD protons showed
appreciable shifts after inclusion complexation with naringenin
(0.01–0.08 ppm). By comparing these chemical shifts, we found
that the shifts of the H-3 protons (0.08 ppm) were larger than
those of the H-5 (0.04 ppm) and H-6 (0.03 ppm) protons, indicat-
ing that naringenin may enter the cavity of TM␤CD from the wide
side as well. It was also revealed that naringenin should penetrate
into the DM␤CD cavity from the wide side (see Supplementary
data).
Two-dimensional (2D) NMR spectroscopy provides impor-
tant information about the spatial proximity between host and
guest atoms via observations of the intermolecular dipolar cross-
correlations (Yang et al., 2009). Two protons that are 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 information, we obtained 2D ROESY of the inclu-
sion complexes of naringenin with CDs. The ROESY spectrum
of the naringenin/␤-CD complex (Fig. 4(a)) showed appreciable
correlation of the H-2ꢀ/H-6ꢀ and H-3ꢀ/H-5ꢀ protons of naringenin
with the H-3, H-5 and H-6 protons of ␤-CD (peaks A). These
results indicate that the C ring of naringenin was included in
3.2. Binding ability
Extensive studies have revealed that the size/shape-fit concept
plays a crucial role in the formation of inclusion complexes of host
CDs 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
inclusion complexation. CDs possess a cyclic truncated cone cav-
ity with a height of 0.79 nm, an inner diameter of 0.62–0.78 nm,
and a cavity volume of 0.262 nm3 for ␤-CD (Ayala-Zavla, Del-Toro-
Sánchez, Alvarez-Parrilla, & González-Aguilar, 2008; Del Valle,
2004; Szejtli, 1998). The host–guest size match may dominate
the stability of the complexes formed between CDs and narin-
genin. From Table 1, we can see that the binding constants for
the complexation of naringenin with ␤-CD, DM␤CD and TM␤CD
were in the following order: ␤-CD > DM␤CD > TM␤CD. By compar-
ing the enhancement effect of all kinds of ␤-CD for naringenin,
␤-CD gave a stronger Ks value than DM␤CD and TM␤CD, which
demonstrated that ␤-CD can complex better with the guest narin-
genin than the methylated CDs. 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 naringenin
may strengthen the host–guest association. In contrast, the methy-
lated CDs (DM␤CD and TM␤CD) showed a weaker Ks value due to
the larger and deeper CD cavity, which caused the intermolecu-
lar hydrogen bond to become weaker (Kano, Nishiyabu, Asada, &
Kuroda, 2002; Reinhardt, Richter, & Mager, 1996).

L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
865
Fig. 3. 1H NMR spectra of naringenin in the absence and presence of ␤-CD, DM␤CD and TM␤CD in D2O at 25 ◦C, respectively. (a) TM␤CD, (b) naringenin/TM␤CD complex,
(c) DM␤CD, (d) naringenin/DM␤CD complex, (e) ␤-CD, and (f) naringenin/␤-CD complex (asterisk highlights the water peak); and (g) the chemical shifts (ı) of the ␤-CD,
TM␤CD, naringenin/␤-CD and naringenin/TM␤CD complexes.
the ␤-CD cavity. The ROESY spectrum of the naringenin/TM␤CD
complex (Fig. 4(b)) also showed significant correlations between
the H-2ꢀ/H-6ꢀ and H-3ꢀ/H-5ꢀ protons of naringenin and the H-
3, H-4, H-5 and H-6 protons of TM␤CD (peaks B). These results
indicate that the C ring of naringenin was also included in the
TM␤CD cavity. It was also shown that naringenin should be
included in the DM␤CD cavity in similar ways (see Supplementary
data).
Based on these observations, together with the 1:1 stoichiom-
etry, we deduced the possible inclusion modes of naringenin with
CDs as illustrated in Fig. 5.
3.4. XRD analysis
The powder X-ray diffraction (XRD) patterns of naringenin,
␤-CD and TM␤CD as well as their inclusion complexes are

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L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
Fig. 4. ROESY spectrum of (a) naringenin/␤-CD complex and (b) naringenin/TM␤CD in D2O.
illustrated in Fig. 6A. As indicated in Fig. 6A, naringenin
(Fig. 6A(a)), ␤-CD (Fig. 6A(b)), DM␤CD (Fig. 6A(c)) and TM␤CD
(Fig. 6A(d)) were in a crystalline form. In contrast, the XRD of
the naringenin/␤-CD, naringenin/DM␤CD and naringenin/TM␤CD
complexes (Fig. 6A(e)–(g)) are amorphous and show halo patterns,
which are quite different from the superimposition of crystalline
naringenin in ␤-CD, DM␤CD and TM␤CD, indicating the formation
of the inclusion complex between ␤-CD (or ␣-CD) and narin-
genin. In addition, most of the crystalline diffraction peaks of ␤-CD,
DM␤CD and TM␤CD disappeared after complexation with narin-
genin, indicating that the complexation of naringenin reoriented
the CD molecules to some extent.
3.5. DSC analysis
The thermal
properties
of
the
naringenin/␤-CD,
naringenin/DM␤CD and naringenin/TM␤CD complexes were
investigated by thermogravimetric (TG) methods (see Supple-
mentary data). A systemic analysis of the TG curves showed
that naringenin decomposes at ca. 210 ◦C, ␤-CD at ca. 200 ◦C,
DM␤CD at ca. 205 ◦C and TM␤CD at ca. 170 ◦C. However, the
thermal stability of their inclusion complexes differed; that is,
the decomposition temperatures were ca. 225, 240 and 245 ◦C for
the naringenin/␤-CD, naringenin/DM␤CD and naringenin/TM␤CD
complexes, respectively. These results indicate that naringenin’s

L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
867
respectively. However, in the DSC curves of the naringenin/CD
complexes, the endothermic peaks at about 255 ◦C corresponding
to the free naringenin disappeared, coinciding with the appearance
of a new exothermic peak at 89, 103, and 105 ◦C in the case of
the naringenin/␤-CD, naringenin/DM␤CD and naringenin/TM␤CD
system (Fig. 6B(e)–(g)), respectively. This suggested that the
naringenin/TM␤CD and naringenin/DM␤CD complexes are more
stable than the naringenin/␤-CD complex.
These results not only further confirm the formation of narin-
genin/CDs complexes, but they also indicate that the naringenin/CD
complexes started to decompose only at a temperature above
225 ◦C, which means that these complexes are fairly stable from
a thermal viewpoint.
H-3
H-5
H
H
6'
O
5'
s
OH
OH
4'
D
HO
C
B
C
2'
A
O
3'
H
H
H-5
H-3
3.6. SEM analysis
Scanning electron microscopy (SEM) is a qualitative method
used to study the structural aspects of raw materials, i.e., CDs and
drugs or the products obtained by different methods of prepara-
tion, such as physical mixing, solution complexation, coevaporation
and others (de Araujo et al., 2008; Duchêne, 1987). The SEM
photographs of DM␤CD, naringenin, their inclusion complexes
and their physical mixtures are shown in Fig. 7. Typical crys-
tals of DM␤CD and naringenin are found in many different
sizes. Pure naringenin crystallizes in a cubic columnar form with
medium dimensions (Fig. 7(a)) and DM␤CD appear as irregu-
larly shaped crystal particles (Fig. 7(b)). The physical mixture of
naringenin/DM␤CD revealed some similarities with the crystals
of the free molecules and showed both crystalline components
(Fig. 7(c)). However, the naringenin/DM␤CD inclusion complex
appeared as a compact and homogeneous plate-like structure crys-
tal and was quite different from the sizes and shapes of ␤-CD
and naringenin (Fig. 7(d)), which confirms the formation of the
naringenin/␤-CD inclusion complex.
naringenin/β-CD complex
naringenin/DMβCD complex
naringenin/TMβCD complex
Fig. 5. Possible inclusion mode and significant NOESY (↔) correlations of the
naringenin/␤-CD, naringenin/DM␤CD and naringenin/TM␤CD inclusion complexes.
usual thermal properties were altered after inclusion complex-
ation, and that the naringenin/CD complexes possessed high
decomposition temperatures.
The differential scanning calorimetry (DSC) thermogram
gave further information about the thermal properties of the
naringenin/␤-CD, naringenin/DM␤CD and naringenin/TM␤CD
complexes. As shown in Fig. 6B, the DSC curve of naringenin
contained an endothermic peak at 255 ◦C (Fig. 6B(a)). In con-
trast, the DSC curves of pristine ␤-CD, DM␤CD and TM␤CD had
an endothermic peak at 118, 68 and 182 ◦C (Fig. 6B(b)–(d)),
A
B
0
5000
-4
-8
a
b
255oC
2500
a
0
10
5
118oC
1600
b
0
0.0
-0.6
-1.2
68oC
7000
c
d
3500
c
0
1200
182oC
21
600
d
14
4
0
89oC
800
e
0
e
400
-4
2
0
800
103oC
0
f
f
400
-2
0
800
105oC
0
-4
-8
g
g
400
0
10
20
40
50
400
2
θ
(degrees)30
50
100
150
200
250
300
350
Temperature/ºC
Fig. 6. (A) XRD patterns: (a) naringenin, (b) ␤-CD, (c) DM␤CD, (d) TM␤CD, (e) naringenin/␤-CD inclusion complex, (f) naringenin/DM␤CD inclusion complex and (g)
naringenin/TM␤CD inclusion complex. (B) DSC thermograms: (a) naringenin, (b) ␤-CD, (c) DM␤CD, (d) TM␤CD, (e) naringenin/␤-CD inclusion complex, (f) naringenin/DM␤CD
inclusion complex and (g) naringenin/TM␤CD inclusion complex.

868
L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
Fig. 7. Scanning electron microphotographs: (a) naringenin, (b) DM␤CD, (c) naringenin and DM␤CD physical mixture (1:1 molar ratio) and (d) naringenin/DM␤CD inclusion
complex.
3.7. Solubilization
and TM␤CD were investigated. The results showed that CDs
and its derivatives can enhance the water solubility and ther-
mal stability of naringenin. Given the shortage of applications for
naringenin and the easy and environmentally friendly prepara-
tion of the naringenin/CD complexes, this inclusion complexation
should be regarded as an important step in the design of a
novel formulation of naringenin for herbal medicine or healthcare
products.
The water solubility of the naringenin/CD complex was assessed
by the preparation of its saturated solution (Montassier, Duchêne,
& Poelman, 1997). An excess amount of the complex was placed
in 2 mL of water (ca. pH 6.0) and the mixture was stirred for
1 h. After removing the insoluble substance by filtration, the fil-
trate was evaporated under reduced pressure to dryness and the
residue was dosed by the weighing method. The results show that
the water solubility of this naringenin, compared to that of native
naringenin (ca. 4.38 ␮g/mL), was remarkably increased to approx-
imately 1.34, 1.60 and 1.52 mg/mL by the solubilizing effects of
␤-CD, DM␤CD and TM␤CD, respectively. In the control experiment,
a clear solution was obtained after dissolving the naringenin/␤-
CD (6.9 mg), naringenin/DM␤CD (9.4 mg) and naringenin/TM␤CD
(9.5 mg) complexes, which was equivalent to 1.34, 1.60 and 1.52 mg
of naringenin, respectively, in 1 mL of water at room temperature.
This confirmed the reliability of the obtained satisfactory water sol-
ubility of the naringenin/CD complex, which will be beneficial for
the medical utilization of this compound.
Acknowledgments
This work was supported by Natural Science Foundation of
China (21162042 and 30960460), Natural Science Foundation of
Yunnan Province (2010CD090 and 2012FB113), Program for Inno-
vative Research Team (in Science and Technology) in University
of Yunnan Province (IRTSTYN) and Program for Teaching Team
of Chemistry Core Curriculum in University of Yunnan Province
(0401-2001019014).
Appendix A. Supplementary data
4. Conclusions
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.carbpol.
2013.07.010.
The inclusion complexation behavior, characterization and
binding ability of naringenin with ␤-CD and its derivatives DM␤CD

L.-J. Yang et al. / Carbohydrate Polymers 98 (2013) 861–869
869
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