Host-guest system of taxifolin and native cyclodextrin or its derivative: Preparation, characterization, inclusion mode, and solubilization

Article abstract of DOI:10.1016/j.carbpol.2011.03.029

The inclusion complexation behavior, characterization and binding ability of taxifolin with native cyclodextrin (α-, β-, or γ-CD) and its derivative hydroxypropyl-β-cyclodextrin (HPβCD) were investigated in both solution and the solid state by means of XR

Full text of DOI:10.1016/j.carbpol.2011.03.029

Carbohydrate Polymers 85 (2011) 629–637
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Host–guest system of taxifolin and native cyclodextrin or its derivative:
Preparation, characterization, inclusion mode, and solubilization
Li-Juan Yanga,b, Wen Chenb, Shui-Xian Mab, Yun-Tao Gaob, Rong Huanga, Sheng-Jiao Yana, Jun Lina,∗
a Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China
b 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
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 taxifolin with native
cyclodextrin (␣-, ␤-, or ␥-CD) and its derivative hydroxypropyl-␤-cyclodextrin (HP␤CD) were inves-
tigated 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 taxifolin were obvi-
ously increased in the inclusion complex with cyclodextrins. This satisfactory water solubility and high
thermal stability of the taxifolin/CD complexes will be potentially useful for their application as herbal
medicines or healthcare products.
Received 6 February 2011
Received in revised form 2 March 2011
Accepted 11 March 2011
Available online 24 March 2011
Keywords:
Taxifolin
Cyclodextrin
Inclusion complex
Characterization
Inclusion mode
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
activity. It has been widely used in the treatment of cerebral infarc-
tion and sequela, cerebral thrombus, coronary heart disease and
Flavonoids are a large group of phenolic compounds and con-
stitute one of the largest groups of secondary metabolites in plants
(Hollman & Katan, 1997; Lin, Mukhopadhyay, Robbins, & Harnly,
2007; Mateus & Costa, 2007; Soares, Mateus, & Freitas, 2007;
Xiao et al., 2008). These substances, with variable polyphenolic
structures, are found in numerous food products, such as fruit,
vegetables, nuts and beverages (coffee, tea, red wine), as well
as in different parts of herbs (Nijeveldt et al., 2001; Shiko et al.,
2009). They are known to possess the ability to scavenge free rad-
icals, and have antimicrobial, antithrombotic, antimutagenic and
anticarcinogenic activities (Belinha et al., 2007; Makris, Boskou, &
Andrikopoulos, 2007; Tu, Lian, Yen, Chen, & Wu, 2007; Wei, Chen,
Jiang, Ma, & Xiao, 2009).
angina pectoris (Landolfi, Mower, & Steiner, 1984; Shiko et al., 2009;
Tzen, Ko, Ko, & Teng, 1991). In recent years, taxifolin has become
a common antioxidant additive in the food industry (Dapkevicius
et al., 2002), and it has also been described as having antimicrobial
activity (Chatzopoulou et al., 2010), anti-inflammatory and anal-
gesic properties (Delporte et al., 2005), anti-adipogenic capacity
(Theriault et al., 2000), radical scavenger activity (Willfor et al.,
2003), and a protective role in plants against pathogens (Bais,
Walker, Kennan, Stermitz, & Vivanco, 2003; Brignolas et al., 1995).
Taxifolin also affects the gastrointestinal tract by possessing antiul-
cer, antispasmodic, and antidiarrheal activities (Di Carlo, Mascolo,
Izzo, & Capasso, 1999). Furthermore, taxifolin has been described
as possessing a tyrosinase inhibitory capacity and thus it is used in
depigmentation drugs and whitening cosmetics, as well as a food
additive and an insect control agent (Miyazawa & Tamura, 2007;
Vega-Villa et al., 2009). Additionally, taxifolin alters the expres-
sion of several genes including those coding for detoxification
enzymes, cell cycle regulatory proteins, growth factors, and DNA
repair proteins (Lee, Cha, Selenge, Solongo, & Nho, 2007). Taxi-
folin is reported to upregulate the phase II detoxification enzymes,
NQO1 and GSTM1, and to downregulate the phase I detoxification
enzyme, CYP2E (Makena, Pierce, Chung, & Sinclair, 2009). How-
ever, the use of taxifolin as a natural antioxidant or herbal medicine
is greatly limited by its low water solubility and bioavailability
(Daniel et al., 2006; Shiko et al., 2009). Although much effort has
been made to improve its water solubility and stability by introduc-
The flavonoid taxifolin (3,5,7,3ꢀ,4ꢀ-pentahydroxyflavanone or
dihydroquercetin, Fig. 1) is widely distributed in barks of the genus
Pinus or Larix and in seeds of the genus Silybum (Daniel, Renaud,
Patrick, & Fabrice, 2006). Several of the plants described as pos-
sessing taxifolin are used in traditional and clinical medicine; some
are ingested in the human diet; and others are being studied for
their potential use in drug development (Vega-Villa et al., 2009).
Taxifolin significantly dilates blood vessels, improves microcircula-
tion, increases cerebral blood flow, and inhibits platelet aggregation
∗
Corresponding author. Tel.: +86 871 503 3215; fax: +86 871 503 3215.
E-mail address: linjun@ynu.edu.cn (J. Lin).
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.03.029

630
L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
OR1
OH
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
n
10 B
3
H-3
H-3
OH
4
5
OH
O
α-CD n=6
β-CD n=7
γ-CD n=8
β-CD
HPβCD R1=R3=H, R2=CH2CH(CH3)OH
R1=R2=R3=H
taxifolin
Fig. 1. The structures of taxifolin, ␣-CD, ␤-CD, ␥-CD and HP␤CD.
ing some nanodispersion or enzymatic condensation techniques
(Daniel, Renaud, Patrick, & Fabrice, 2009; Shiko et al., 2009), it is
still not possible to sufficiently dissolve taxifolin in water, which
prevents its usage for some therapeutic and cosmetic applica-
tions. Therefore, the search for an efficient and nontoxic carrier
for taxifolin has become important in order to further its clinical
applications.
It is well known that cyclodextrins (CDs) are truncated-
cone polysaccharides mainly composed of six to eight d-glucose
monomerslinked by ␣-1,4-glucosebonds. They haveahydrophobic
central cavity and a 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 solutions and affects the chemical characteristics 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), and photochemical
sensors (Ueno, 1996), etc. Recently, some studies of inclusion com-
plex 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). However, to the
best of our knowledge, no scientific study on the inclusion behavior
of taxifolin/CD complexes has hitherto been reported.
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, 2009a), scutellarin (Yang, Yang,
Lin, Chen, & Liu, 2009b), azadirachtin B (Yang, Chen, Lin, & Liu,
2008; Yang & Lin, 2009) and crassicauline A (Chen et al., 2011),
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, Lin, et al., 2009a;
Yang, Yang, et al., 2009b; Yang et al., 2010). As a continuation of
0.8
0.04
0.6
0.012
0.7
Exp
g
a
h
a
0.008
0.004
0.000
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.5
0.4
0.3
0.2
0.1
0.0
0.02
Calcd
Exp.
Calcd.
0.00
0.0
0.4
0.8
1.2
1.6
0
1
2
3
4
5
[γ-CD]/mM
[β-CD] / mM
250
300
350
400
250
300
350
400
λ/nm
2
λ/nm
1
0.5
0.4
0.3
0.2
0.1
0.0
0.015
0.010
0.005
0.000
g
Exp.
Calcd.
a
0.0 0.5 1.0 1.5 2.0 2.5
[α -CD] / mM
250
300
350
400
3
λ/nm
Fig. 2. UV–vis spectral changes in taxifolin (0.03 mM) upon addition of ␤-CD (1: 0–1.50 mM, from a to h), ␥-CD (2: 0–4.20 mM, from a to g) and ␣-CD (3: 0–2.60 mM, from
a to g) 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 287 nm) to calculate the
complex stability constant (Ks).

L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
631
*
H-5,6
H-1
H-3
a
b
*
*
H-5,6
H-1
H-3
c
d
*
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 0.5 ppm
δ (ppm)
e
β-CD
4.99
3.57
3.88
3.50
3.80
3.78
taxifolin/β-CD complex
HPβCD
4.99
3.54
3.87
3.49
3.77
3.77
1.03
taxifolin/HPβCD complex
H-1
H-2
H-3
H-4
H-5
H-6
H-Me
d
5.04
3.62
3.91
3.56
3.81
3.76
5.02
3.57
3.97
3.45
3.78
3.75
1.13
dd
dd
dd
m
dd
s
Fig. 3. 1H NMR spectra of taxifolin in the absence and presence of ␤-CD and HP␤CD in D2O at 25 ◦C, respectively. (a) ␤-CD, (b) taxifolin/␤-CD complex, (c) HP␤CD, (d)
taxifolin/HP␤CD complex (asterisk highlights the water peak); and the chemical shifts (ı) of the ␤-CD, HP␤CD, taxifolin/␤-CD and taxifolin/HP␤CD complexes (e).
our studies on natural medicines/cyclodextrin inclusion complex,
an inclusion complex of CLA with ␤-CD was investigated.
In this paper, we aim to report the preparation and charac-
terization of some water-soluble inclusion complexes formed by
taxifolin and native cyclodextrin (␣-, ␤-, or ␥-CD) and its deriva-
tive: 2-hydroxypropyl-␤-cyclodextrin (HP␤CD) (Fig. 1). We were
particularly interested in exploring the solubilization effect of CDs
on taxifolin and the binding ability of the resulting inclusion com-
plexes, which would provide a useful approach for obtaining novel
taxifolin-basedhealthcareproductswithhighwatersolubility, high
bioavailability and low toxicity.
␥-CD (average substitution degree = 1297), and 2-hydroxypropyl-
␤-cyclodextrin (HP␤CD, average substitution degree = 1380) were
purchased from ABCR GmbH and 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. Methods
2.2.1. Preparation of taxifolin/ˇ-CD, taxifolin/HPˇCD,
taxifolin/˛-CD and taxifolin/ꢁ-CD complexes
Taxifolin (0.03 mM, 9.1 mg) and CD (0.01 mM) were completely
dissolved in a mixed solution of ethanol and water (ca. 7 mL,
V:V = 1:5, given the poor water solubility of taxifolin, ethanol was
used), and the mixture was stirred for 5 days at room temper-
ature. After evaporating the ethanol from the reaction mixture,
the uncomplexed taxifolin was removed by filtration. The filtrate
was evaporated under reduced pressure to remove the solvent
and dried in vacuum to produce the taxifolin/CDs complexes.
Taxifolin/␤-CD complex (yield 92%): 1H NMR (500 MHz, D2O, TMS):
ı 6.85–7.05 (m, 3H, H-2ꢀ, 5ꢀ, 6ꢀ of C ring protons for taxifolin),
3.45–3.95 (m, 42H, H-2–6 of ␤-CD), 4.97–4.99 (s, 7H, H-1 of ␤-
CD). Taxifolin/HP␤CD complex (yield 91%): 1H NMR (500 MHz, D2O,
TMS): ı 6.85–7.05 (m, 3H, H-2ꢀ, 5ꢀ, 6ꢀ of C ring protons for taxifolin),
3.30–4.15 (m, 100H, H-2–6 and CH2- and CH3-2, 3, 6 of HP␤CD),
4.95–5.10 (s, 7H, H-1 of HP␤CD). Taxifolin/␣-CD complex (yield
80%): 1H NMR (500 MHz, D2O, TMS): ı 6.85–7.05 (m, 3H, H-2ꢀ, 5ꢀ,
6ꢀ of C ring protons for taxifolin), 3.45–4.00 (m, 36H, H-2–6 of ␣-
2. Materials and methods
2.1. Materials
Taxifolin (FW = 304, PC > 98%) was obtained from Nanjing Zelang
Medical Technology Co., Ltd. (Jiangu, PR China); ␣-CD (average sub-
stitution degree = 972), ␤-CD (average substitution degree = 1135),
Table 1
The stability constant (Ks and log Ks) and Gibbs free energy change (−ꢀG◦) for the
inclusion complexation of CDs with taxifolin guest in a water/alcohol (V:V = 4:1, ca.
pH 3.0) mixed solution.
Host
Ks/M−1
log Ks
−ꢀG◦/kJ mol−1
␣-CD
␤-CD
␥-CD
1872
2145
2908
3.27
3.33
3.46
18.68
19.02
19.77

632
L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
CD), 4.90–5.05 (s, 7H, H-1 of ␣-CD). Taxifolin/␥-CD complex (yield
82%): 1H NMR (500 MHz, D2O, TMS): ı 6.85–7.05 (m, 3H, H-2ꢀ, 5ꢀ, 6ꢀ
of C ring protons for taxifolin), 3.40–3.85 (m, 56H, H-2–6 of ␥-CD),
4.94–5.00 (s, 7H, H-1 of ␥-CD).
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.
2.2.2. Spectral titration
3. Results and discussion
Absorption spectra measurements were carried out with a
Shimadzu UV 2401 (Japan) using
a conventional 1 cm path
3.1. Spectral titration
(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 taxifolin, a water/ethanol
(V:V = 4:1) solution was used in the spectral measurements. The
concentration of taxifolin was held constant at 0.05 mM. Then, an
appropriate amount of CD was added, and the final concentrations
varied from 0 to 1.50–4.20 mM (␤-CD: 0, 0.06, 0.13, 0.26, 0.52, 0.74,
1.06, 1.50 mM; ␥-CD: 0, 0.25, 0.50, 1.01, 1.44, 2.97, 4.20 mM; ␣-
CD: 0, 0.06, 0.22, 0.45, 0.89, 1.27, 2.60 mM). The absorption 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.
A quantitative investigation of the inclusion complexation
behavior of ␣-, ␤-, ␥-CDs and HP␤CD with taxifolin 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 taxifolin. As illustrated in Fig. 2, the absorbance intensity of
taxifolin gradually increased with the stepwise addition of ␤-CD,
␥-CD and ␣-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 controlling factors on
the formation of inclusion complexes of CDs (Liu & Chen, 2006),
these results indicate that the binding behavior is mainly depen-
dent on the individual structural features of the host and guest.
Assuming a 1:1 stoichiometry for the taxifolin/CD inclusion com-
plex, the inclusion complexation of taxifolin with ␤-CD could be
expressed by Eq. (1), and the stability constant (Ks) could be calcu-
lated from Eq. (2), where [taxifolin/CD], [taxifolin], [CD], [taxifolin]0
and [CD]0 refer to the equilibrium concentration of the taxifolin/CD
inclusion complex, the equilibrium concentration of taxifolin, the
equilibrium concentration of CD, the original concentration of tax-
ifolin, and the original concentration of CD, respectively, and ꢀε
is the differential molar extinction coefficient of taxifolin in the
absence and presence of CD. According to Lambert–Beer Law, it
was found that the concentration of the taxifolin/CD complex was
equal to ꢀ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
Eq. (3).
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.
2.2.4. Powder X-ray diffraction (XRD)
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 2ꢂ = 0.02◦ between 2ꢂ = 3◦ and 50◦.
K
s
Taxifolin + CDꢀTaxifolin · CD
(1)
[Taxifolin · CD]
ꢀA/ꢀε
Ks =
=
2.2.5. Thermal analyses
Differential scanning calorimetry (DSC) and thermogravimet-
ric (TG) measurements were performed with a 2960 SDT V3.0F
[Taxifolin][CD]
([Taxifolin]0 − ꢀA/ꢀε)([CD]0 − ꢀA/ꢀε)
(2)
ꢀ
ꢀε([Taxifolin]0 + [CD]0 + 1/Ks)
ꢀε2([Taxifolin]0 + [CD]0 + 1/Ks)2 − 4ꢀε2[Taxifolin]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 typical
curve-fitting plot for the titration of taxifolin with ␤-CD, ␥-CD and
␣-CD, which shows the excellent fit between the experimental and
calculated data and the 1:1 stoichiometry of the taxifolin/CDs inclu-
sion complexes. In the 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
CDs with taxifolin are listed in Table 1.
instrument and NETZSCH STA 449F3, respectively, at the a heating
rate of 10 ◦C/min from room temperature to 400 ◦C in a dynamic
nitrogen atmosphere (flow rate = 70 mL/min).
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
3.2. Binding ability
15, 20 or 30 kV and a magnification of 1080, 1200, 1400 or 2000×.
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,
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

L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
633
Fig. 4. ROESY spectrum of (a) taxifolin/␤-CD complex and (b) taxifolin/HP␤CD complex in D2O.
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
cavity with a height of 0.79 nm, an inner diameter of 0.47–0.53,
0.62–0.78 and 0.75–0.83 nm, and a cavity volume of 0.174, 0.262
and 0.427 nm3 for the ␣-, ␤- and ␥-CDs, respectively (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 taxifolin.
From Table 1, we can see that the binding constants for the com-
plexation of taxifolin with ␣-, ␤- and ␥-CDs were in the following
order: ␥-CD > ␤-CD > ␣-CD. By comparing the enhancement effect
of all kinds of native CDs for taxifolin, ␥-CD and ␤-CD gave a
stronger Ks value than ␣-CD, which showed that ␥-CD and ␤-CD,
which possessed a larger cavity size, can complex better with the
guest taxifolin than ␣-CD. It was showed that the size-fit effect
was the dominant controlling factor on the formation of inclusion
complexes of taxifolin/CDs.

634
L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
To further explore the inclusion mode, the chemical shifts of ␤-
CD protons in the absence and presence of taxifolin were examined
(Fig. 3). Inclusion complexation with taxifolin had 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 (0.03–0.06 ppm),
which could have been caused by the hydrogen bond between
the hydroxyl arms of ␤-CD and the oxygen atoms of taxifolin. It
is noteworthy that the H-3 protons 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 taxifolin should penetrate into the
␤-CD cavity from the wide side. Similarly, all of the HP␤CD pro-
tons showed appreciable shifts after inclusion complexation with
taxifolin (0.01–0.10 ppm). By comparing these chemical shifts, we
found that the shifts of the H-3 protons (0.10 ppm) were larger than
those of the H-5 (0.01 ppm) and H-6 (0.02 ppm) protons, indicating
that taxifolin may enter the cavity of HP␤CD from the wide side
as well. It was also revealed that taxifolin should penetrate into
the ␣-CD and ␥-CD cavities from the wide side (see Supplementary
data).
H-3
H-5
H-5
HO
H
5'
H
6'
O
OH
OH
HO
C
B
2'
A
O
HO
H
H-3
taxifolin/α-CD complex
taxifolin/γ-CD complex
taxifolin/β-CD complex
taxifolin/HPβCD complex
Fig. 5. Possible inclusion mode and significant NOESY (↔) correlations
of the taxifolin/␤-CD, taxifolin/HP␤CD, taxifolin/␣-CD and taxifolin/␥-CD
inclusion complexes.
3.3. 1H and 2D NMR analysis
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, Lin, et al., 2009a; Yang, Yang, et al., 2009b). 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 pro-
tons 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 inclusion complexes of taxifolin with CDs.
The ROESY spectrum of the taxifolin/CD complex (Fig. 4a) showed
appreciable correlation of the H-2ꢀ, H-5ꢀ and H-6ꢀ protons of tax-
ifolin with the H-3, H-5 and H-6 protons of ␤-CD (peak a). These
results indicate that the C ring of taxifolin was included in the ␤-
CD cavity. The ROESY spectrum of the taxifolin/HP␤CD complex
In order to explore the possible inclusion mode of the taxi-
folin/CD complex, we compared the 1H NMR spectra of taxifolin
in the presence of the host CDs (Fig. 3). Owing to its poor water sol-
ubility, taxifolin is transparent to 1H NMR under most conditions
when D2O is used as a solvent. Assessment of the taxifolin complex
by 1H NMR clearly demonstrated the presence of the framework
protons of the taxifolin molecule, consistent with the significant
solubilization. As illustrated in Fig. 3, the majority of taxifolin pro-
tons displayed chemical shifts at ı 5.0–7.5 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 CDs H-1 pro-
tons, we calculated the inclusion stoichiometry of the taxifolin/CD
complexes, that is, 1:1 for the taxifolin/␤-CD and taxifolin/HP␤CD
complexes. This 1:1 inclusion stoichiometry was also observed in
the taxifolin/␣-CD and taxifolin/␥-CD complexes.
Fig. 6. (A) XRD patterns: (a) taxifolin, (b) ␤-CD, (c) ␣-CD, (d) taxifolin/␤-CD inclusion complex, (e) taxifolin/␣-CD inclusion complex; (B) DSC thermograms: (a) taxifolin, (b)
␤-CD, (c) HP␤CD, (d) ␥-CD, (e) taxifolin/␤-CD inclusion complex, (f) taxifolin/HP␤CD inclusion complex, (g) taxifolin/␥-CD inclusion complex.

L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
635
Fig. 7. (A) Scanning electron microphotographs: (a) taxifolin, (b) ␤-CD, (c) taxifolin ␤-CD physical mixture (1:1 molar ratio), (d) taxifolin/␤-CD inclusion complex; (B)
Scanning electron microphotographs: (a) taxifolin, (b) HP␤CD, (c) taxifolin and HP␤CD physical mixture (1:1 molar ratio), (d) taxifolin/HP␤CD inclusion complex.
(Fig. 4b) also showed significant correlations between the H-2ꢀ, H-
5ꢀ and H-6ꢀ protons of taxifolin and the H-3, H-5 and H-6 protons of
HP␤CD (peaks b). These results indicate that the C ring of taxifolin
was also included in the HP␤CD cavity. It was also shown that tax-
ifolin should be included in the ␣-CD and ␥-CD cavities in similar
ways (see Supplementary data).
Based on these observations, together with the 1:1 stoichiome-
try, we deduced the possible inclusion modes of taxifolin with CDs
as illustrated in Fig. 5.
3.4. XRD analysis
The powder X-ray diffraction (XRD) patterns of taxifolin, ␤-CD
and ␣-CD as well as their inclusion complexes are illustrated in
Fig. 6A (the XRD of HP␤CD and ␥-CD and their inclusion com-
plexes with taxifolin can be seen in the Supplementary data). As
indicated in Fig. 6A, taxifolin (Fig. 6A(a)), ␤-CD (Fig. 6A(b)) and ␣-
CD (Fig. 6A(c)) were in a crystalline form. In contrast, the XRD of
the taxifolin/␤-CD and taxifolin/␣-CD complexes (Fig. 6A(d) and

636
L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
(e)) are amorphous and show halo patterns, which are quite differ-
ent from the superimposition of crystalline taxifolin in ␤-CD and
␣-CD, indicating the formation of the inclusion complex between
␤-CD (or ␣-CD) and taxifolin. A similar phenomenon for HP␤CD and
␥-CD and their inclusion complexes was found (see Supplementary
data). In addition, most of the crystalline diffraction peaks of ␤-CD,
␣-CD, HP␤CD and ␥-CD disappeared after complexation with tax-
ifolin, indicating that the complexation of taxifolin reoriented the
CD molecules to some extent.
(Fig. 7B(d)), which confirms the formation of the taxifolin/HP␤CD
inclusion complex.
3.7. Solubilization
The water solubility of the taxifolin/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 filtrate
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 taxifolin, compared to that of native taxi-
folin (ca. 1.0 mg/mL), was remarkably increased to approximately
12.1, 6.8, 8.5, and 7.0 mg/mL by the solubilizing effects of ␤-CD,
HP␤CD, ␣-CD, and ␥-CD, respectively. In the control experiment,
a clear solution was obtained after dissolving the taxifolin/␤-CD
(23.8 mg), taxifolin/HP␤CD (18.0 mg), taxifolin/␣-CD (22.0 mg), and
taxifolin/␥-CD (20.0 mg) complexes, which was equivalent to 12.1,
6.8, 8.5, and 7.0 mg of taxifolin, respectively, in 1 mL of water at
room temperature. This confirmed the reliability of the obtained
satisfactory water solubility of the taxifolin/CD complex, which will
be beneficial for the medical utilization of this compound.
3.5. DSC analysis
The thermal properties of the taxifolin/␤-CD, taxifolin/HP␤CD
and taxifolin/␥-CD complexes were investigated by thermogravi-
metric (TG) methods (see Supplementary data). A systemic analysis
of the TG curves showed that taxifolin decomposes at ca. 190 ◦C,
␤-CD at ca. 200 ◦C, ␥-CD at ca. 208 ◦C and HP␤CD at ca. 210 ◦C. How-
ever, the thermal stability of their inclusion complexes differed;
that is, the decomposition temperatures were ca. 230, 220 and
250 ◦C for the taxifolin/␤-CD, taxifolin/␥-CD and taxifolin/HP␤CD
complexes, respectively. These results indicate that taxifolin’s usual
thermal properties were altered after inclusion complexation, and
that the taxifolin/CD complexes possessed high decomposition
temperatures.
The differential scanning calorimetry (DSC) thermogram gave
further information about the thermal properties of the taxifolin/␤-
CD, taxifolin/HP␤CD and taxifolin/␥-CD complexes. As shown in
Fig. 6B, the DSC curve of taxifolin contained an endothermic peak
at 130 ◦C (Fig. 6B(a)). In contrast, the DSC curves of pristine ␤-CD,
HP␤CD and ␥-CD had an endothermic peak at 99, 83 and 88 ◦C
(Fig. 6B(b)–(d)), respectively. However, in the DSC curves of the
taxifolin/CD complexes, the endothermic peaks at about 130 ◦C
corresponding to the free taxifolin disappeared, coinciding with
the appearance of a new exothermic peak at 76, 85 and 83 ◦C in
the case of the taxifolin/␤-CD, taxifolin/HP␤CD and taxifolin/␥-
CD system (Fig. 6B(e)–(g)), respectively. This suggested that the
taxifolin/HP␤CD complex was more stable than the taxifolin/␤-CD
or taxifolin/␥-CD complexes.
4. Conclusions
The inclusion complexation behavior, characterization and
binding ability of taxifolin with native cyclodextrin (␣-, ␤-, or
␥-CD) and its derivative hydroxypropyl-␤-cyclodextrin (HP␤CD)
were investigated. The results showed that CDs and its derivatives
can enhance the water solubility and thermal stability of taxifolin.
Given the shortage of applications for taxifolin and the easy and
environmentally friendly preparation of the taxifolin/CD complex,
this inclusion complexation should be regarded as an important
step in the design of a novel formulation of taxifolin for herbal
medicine or healthcare products.
Acknowledgements
These results not only further confirm the formation of taxi-
folin/CDs complexes, but they also indicate that the taxifolin/CD
complexes started to decompose only at a temperature above
220 ◦C, which means that these complexes are fairly stable from
a thermal viewpoint.
This work was supported by NSFC (30860342), Natural Sci-
ence Foundation of Yunnan Province (2010CD090, 2009cc017),
the Opening Foundation of the State Key Laboratory of Elemento-
Organic Chemistry of Nankai University (0704 and 0815), and
Natural Science Foundation of Yunnan Education Department
(2010Y429).
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 pho-
tographs of ␤-CD, taxifolin, their inclusion complexes and their
physical mixtures are shown in Fig. 7A. Typical crystals of ␤-CD and
CLA are found in many different sizes. Pure taxifolin appear as irreg-
ularly shaped crystal particles (Fig. 7A(a)) and ␤-CD crystallizes in
a polyhedral form with large dimensions (Fig. 7A(b)). The physi-
cal mixture of taxifolin/␤-CD revealed some similarities with the
crystals of the free molecules and showed both crystalline compo-
nents (Fig. 7A(c)). However, the taxifolin/␤-CD inclusion complex
appeared as a compact and homogeneous plate-like structure with
crystal particles and was quite different from the sizes and shapes
of ␤-CD and taxifolin (Fig. 7A(d)), which confirms the formation
of the taxifolin/␤-CD inclusion complex. In similar tests, HP␤CD
appeared as a spherical crystal with cavity structures (Fig. 7B(b)).
However, the taxifolin/HP␤CD inclusion complex appeared to be
quite different from the sizes and shapes of HP␤CD and taxifolin
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carbpol.2011.03.029.
References
Ayala-Zavla, J. F., Del-Toro-Sánchez, L., Alvarez-Parrilla, E., & González-Aguilar, G.
A. (2008). High relative humidity in-package of fresh-cut fruits and vegetables:
Advantage or disadvantage considering microbiological problems and antimi-
crobial delivering systems? Journal of Food Science, 73, R41–R47.
Bais, H. P., Walker, T. S., Kennan, A. J., Stermitz, F. R., & Vivanco, J. M. (2003).
Structure-dependent phytotoxicity of catechins and other flavonoids: Flavonoid
conversions by cell-free protein extracts of Centaurea maculosa (spotted knap-
weed) roots. Journal of Agricultural and Food Chemistry, 51, 897–901.
Belinha, I., Amorim, M. A., Rodrigues, P., de Freitas, V., Moradas-Ferreira, P., & Japon-
Lujan, R. (2007). Static-dynamic superheated liquid extraction of hydroxytyrosol
and other biophenols from Alperujo (a semisolid residue of the olive oil indus-
try). Journal of Agricultural and Food Chemistry, 55, 3629–3634.
Bian, H., Chen, J., Cai, X., Liu, P., Liu, H., Qiao, X., et al. (2009). Inclusion complex
of butachlor with ␤-cyclodextrin: Characterization solubility, and speciation-
dependent adsorption. Journal of Agricultural and Food Chemistry, 57, 7458–7543.

L.-J. Yang et al. / Carbohydrate Polymers 85 (2011) 629–637
637
Breslow, R., & Dong, S. D. (1998). Biomimetic reactions catalyzed by cyclodextrins
and their derivatives. Chemical Reviews, 98, 1997–2012.
Brignolas, F., Lacroix, B., Lieutier, F., Sauvard, D., Drouet, A., Claudot, A. C., et al.
(1995). Induced responses in phenolic metabolism in two Norway spruce clones
after wounding and inoculations with Ophiostoma polonicum, a bark beetle-
associated fungus. Plant Physiology, 109, 821–827.
Misiuk, W., & Zalewska, M. (2009). Investigation of inclusion complex of trazodone
hydrochloride with hydroxypropyl-␤-cyclodextrin. Carbohydrate Polymers, 77,
482–488.
Miyazawa, M., & Tamura, N. (2007). Inhibitory compound of tyrosinase activity from
the sprout of Polygonum hydropiper L. (Benitade). Biological & Pharmaceutical
Bulletin, 30, 595–597.
Cannavà, C., Crupib, V., Ficarra, P., Guardo, M., Majolino, D., Mazzaglia, A., et al. (2010).
Physico-chemical characterization of an amphiphilic cyclodextrin/genistein
complex. Journal of Pharmaceutical and Biomedical Analysis, 51, 1064–1068.
Chatzopoulou, A., Karioti, A., Gousiadou, C., Vivancos, V. L., Kyriazopoulos, P., Gole-
gou, S., et al. (2010). Depsides and other polar constituents from Origanum
dictamnus L. and their in vitro antimicrobial activity in clinical strains. Journal of
Agricultural and Food Chemistry, 58, 6064–6068.
Montassier, P., Duchêne, D., & Poelman, M. C. (1997). Inclusion complexes of tretinoin
with cyclodextrins. International Journal of Pharmaceutics, 153, 199–209.
Mourtzinos, I., Makris, D. P., Yannakopoulou, K., Kalogeropoulos, N., Michali, I., &
Karathanos, V. T. (2008). Thermal stability of anthocyanin extract of Hibiscus
sabdariffa L. in the presence of ␤-cyclodextrin. Journal of Agricultural and Food
Chemistry, 56, 10303–10310.
Nijeveldt, R. J., van Nood, E., van Hoorn, D. E. C., Boelens, P. G., van Norren, K., & van
Leeuwen, P. A. M. (2001). Flavonoids: A review of probable mechanisms of action
and potential applications. American Journal of Clinical Nutrition, 74, 418–425.
Shiko, A. N., Pozharitskaya, O. N., Miroshnyk, I., Mirzab, S., Urakova, I. N., Hirsjävi, S.,
et al. (2009). Nanodispersions of taxifolin: Impact of solid-state properties on
dissolution behavior. International Journal of Pharmaceutics, 377, 148–152.
Soares, S., Mateus, N., & Freitas, V. (2007). Interaction of different polyphenols with
bovine serum albumin (BSA) and human salivary r-amylase (HSA) by fluores-
cence quenching. Journal of Agricultural and Food Chemistry, 55, 6726–6735.
Szejtli, J. (1998). Introduction and general overview of cyclodextrin chemistry. Chem-
ical Reviews, 98, 1743–1753.
Theriault, A., Wang, Q., Van Iderstine, S. C., Chen, B., Franke, A. A., & Adeli, K. (2000).
Modulation of hepatic lipoprotein synthesis and secretion by taxifolin, a plant
flavonoid,. Journal of Lipid Research, 41, 1969–1979.
Tommasini, S, Raneri, D., Ficarra, R., Calabrò, M. L., Stancanelli, R., & Ficarra, P.
(2004). Improvement in solubility and dissolution rate of flavonoids by complex-
ation with ␤-cyclodextrin,. Journal of Pharmaceutical and Biomedical Analysis, 35,
379–387.
Chen, W., Yang, L. J., Ma, S. X., Yang, X. D., Fan, B. M.,
& Lin, J. (2011).
Crassicauline A/␤-cyclodextrin host–guest system: Preparation, characteriza-
tion, inclusion mode, solubilization and stability. Carbohydrate Polymers, 83
doi:10.1016/j.carbpol.2011.01.031
Correia, I., Bezzenine, N., Ronzani, N., Platzer, N., Beloeil, J.-C., & Doan, B.-T. (2002).
Study of inclusion complexes of acridine with ␤- and (2,6-di-O-methyl)-␤-
cyclodextrin by use of solubility diagrams and NMR spectroscopy. Journal of
Physical Organic Chemistry, 15, 647–659.
Daniel, A., Renaud, N., Patrick, R., & Fabrice, L. (2006). Water soluble phenolics
derivatives with dermocosmetic and therapeutic applications. European Patent,
EP1867729.
Daniel, A., Renaud, N., Patrick, R., & Fabrice, L. (2009). Water soluble and activable
phenolics derivatives with dermocosmetic and therapeutic applications and
process for preparing said derivatives. United States Patent, US20090233876.
Dapkevicius, A., van Beek, T. A., Lelyveld, G. P., van Veldhuizen, A., de Groot, A.,
Linssen, J. P., et al. (2002). Isolation and structure elucidation of radical scav-
engers from Thymus vulgaris leaves. Journal of Natural Products, 65, 892–896.
de Araujo, D. R., Tsuneda, S. S., Cereda, C. M. S., Carvalho, F. D. G. F., Preté, P. S.
C., Fernandes, S., et al. (2008). Development and pharmacological evaluation
of ropivacaine-2-hydroxypropyl-␤-cyclodextrin inclusion complex. European
Journal of Pharmaceutical Sciences, 33, 60–71.
Tu, Y., Lian, T., Yen, J., Chen, Z., & Wu, M. (2007). Antiatherogenic effects of kaempferol
and rhamnocitrin. Journal of Agricultural and Food Chemistry, 55, 9969–9976.
Tzen, S. H., Ko, W., Ko, F., & Teng, C. (1991). Inhibition of platelet aggregation by some
flavonoids. Thrombosis Research, 64, 91–100.
Del Valle, E. M. M. (2004). Cyclodextrins and their uses: A review. Process Biochem-
istry, 39, 1033–1046.
Uekama, K., Hirayama, F., & Irie, T. (1998). Cyclodextrin drug carrier systems. Chem-
ical Reviews, 98, 2045–2076.
Delporte, C., Backhouse, N., Erazo, S., Negrete, R., Vidal, P., Silva, X., et al.
(2005). Analgesic-antiinflammatory properties of Proustia pyrifolia. Journal of
Ethnopharmacology, 99, 119–124.
Ueno, A. (1996). Review: Fluorescent cyclodextrins for molecule sensing.
Supramolecular Science, 3, 31–36.
Vega-Villa, K. R., Remsberg, C. M., Ohgami, Y., Yán´ ez, J. A., Takemoto, J. K., Andrews, P.
K., et al. (2009). Stereospecific high-performance liquid chromatography of taxi-
folin, applications in pharmacokinetics, and determination in tu fu ling (Rhizoma
smilacis glabrae) and apple (Malus × domestica). Biomedical Chromatography, 23,
638–646.
Wang, J., & Cai, Z. (2008). Investigation of inclusion complex of miconazole nitrate
with ␤-cyclodextrin. Carbohydrate Polymers, 72, 255–260.
Wei, Y., Chen, X., Jiang, X., Ma, Z., & Xiao, J. (2009). Determination of taxifolin in
Polygonum orientale and study on its antioxidant activity. Journal of Food Com-
position and Analysis, 22, 154–157.
Di Carlo, G., Mascolo, N., Izzo, A. A., & Capasso, F. (1999). Flavonoids: Old and new
aspects of a class of natural therapeutic drugs. Life Science, 65, 337–353.
Duchêne, D. (1987). Cyclodextrins and their industrial uses. Paris: Editions de Santé.
Hollman, P. C., & Katan, M. B. (1997). Absorption, metabolism, and health effects of
dietary flavonoids in man. Biochemical Pharmacology, 51, 305–310.
Koontz, J. L., Marcy, J. E., O’Keefe, S. F., & Duncan, S. E. (2009). Cyclodextrin inclusion
complex formation and solid-state characterization of the natural antioxidants
␣-tocopherol and quercetin. Journal of Agricultural and Food Chemistry, 57,
1162–1171.
Landolfi, R., Mower, R. L., & Steiner, M. (1984). Modification of platelet function
and arachidonic acid metabolism by bioflavonoids. Structure–activity relations.
Biochemical Pharmacology, 33, 1525–1530.
Lee, S. B., Cha, K. H., Selenge, D., Solongo, A., & Nho, C. W. (2007). The chemopre-
ventive effect of taxifolin is exerted through ARE-dependent gene regulation.
Biological & Pharmaceutical Bulletin, 30, 1074–1079.
Lin, L. Z., Mukhopadhyay, S., Robbins, R. J., & Harnly, J. M. (2007). Identification and
quantification of flavonoids of Mexican oregano (Lippia graveolens) by LC-DAD-
ESI/MS analysis. Journal of Food Composition and Analysis, 20, 361–369.
Liu, Y., & Chen, Y. (2006). Cooperative binding and multiple recognition by bridged
bis(␤-cyclodextrin)s with functional linkers. Accounts of Chemical Research, 39,
681–691.
Liu, Y., Li, B., Wada, T., & Inoue, Y. (1999). Novel o-phenylenediseleno bridged
bis (␤-cyclodextrin)s complexes with platinum(IV) and palladium(II) ions.
Supramolecular Chemistry, 10, 279–285.
Willfor, S. M., Ahotupa, M. O., Hemming, J. E., Reunanen, M. H., Eklund, P. C., Sjoholm,
R. E., et al. (2003). Antioxidant activity of knotwood extractives and phenolic
compounds of selected tree species. Journal of Agricultural and Food Chemistry,
51, 7600–7606.
Wu, H., Liang, H., Yuan, Q., Wang, T., & Yan, X. (2010). Preparation and stability
investigation of the inclusion complex of sulforaphane with hydroxypropyl-␤-
cyclodextrin. Carbohydrate Polymers, 82, 613–617.
Xiao, J. B., Suzuki, M., Jiang, X. Y., Chen, X. Q., Yamamoto, K., & Xu, M. (2008). Influence
of B-ring hydroxylation on interactions of flavonols with bovine serum albumin.
Journal of Agricultural and Food Chemistry, 56, 2350–2356.
Yang, B., Chen, Y., Lin, J., & Liu, Y. (2008). Inclusion complexation behaviors of
3-tigloyl-azadirachtol with ␤-cyclodextrin derivatives. Current Pharmaceutical
Analysis, 4, 176–182.
Yang, B., & Lin, J. (2009). Selective binding behaviors of ␤-cyclodextrin and its deriva-
tives with azadirachtin guests by competitive inclusion method. Chinese Journal
of Analytical Chemistry, 37, 1468–1472.
Yang, B., Lin, J., Chen, Y., & Liu, Y. (2009). Artemether/hydroxypropyl-␤-cyclodextrin
host–guest system: Characterization, phase-solubility and inclusion mode.
Bioorganic and Medicinal Chemistry, 17, 6311–6317.
Makena, P. S., Pierce, S. C., Chung, K. T., & Sinclair, S. E. (2009). Comparative mutagenic
effects of structurally similar flavonoids quercetin and taxifolin on tester strains
Salmonella typhimurium TA102 and Escherichia coli WP-2 uvrA. Environmental
and Molecular Mutagenesis, 50, 451–459.
Makris, D. P., Boskou, G., & Andrikopoulos, N. K. (2007). Polyphenolic content and
in vitro antioxidant characteristics of wine industry and other agri-food solid
waste extracts. Journal of Food Composition and Analysis, 20, 125–132.
Mateus, N., & Costa, V. (2007). Quercetin increases oxidative stress resistance and
longevity in Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry,
55, 2446–2451.
Yang, B., Yang, L. J., Lin, J., Chen, Y., & Liu, Y. (2009). Binding behaviors of scutellarin
with ˛-, ˇ-, ꢁ-cyclodextrins and their derivatives. Journal of Inclusion Phenomena
and Molecular Recognition in Chemistry, 64, 149–155.
Yang, L. J., Yang, B., Chen, W., Huang, R., Yan, S. J., & Lin, J. (2010). Host–guest system
of nimbin and ␤-cyclodextrin or its derivatives: Preparation, characterization,
inclusion mode, and solubilization. Journal of Agricultural and Food Chemistry,
58, 8545–8552.