Inclusion complexes of phosphorylated daidzein derivatives with β-cyclodextrin: Preparation and inclusion behavior study

Article abstract of DOI:10.1016/j.saa.2011.10.014

In the present work the feasibility of β-cyclodextrin in complexation was explored, as a tool for improving the solubility and biological ability of daidzein derivatives. A series of phosphorylated daidzein derivatives featuring different chain lengths we

Full text of DOI:10.1016/j.saa.2011.10.014

Spectrochimica Acta Part A 85 (2012) 298–302
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Inclusion complexes of phosphorylated daidzein derivatives with
␤-cyclodextrin: Preparation and inclusion behavior study
Yongmei Xiao∗, Liangru Yang, Pu Mao, Jinwei Yuan, Yuxia Deng, Lingbo Qu
College of Chemistry & Chemical Engineering, Henan University of Technology, Zhengzhou 450001, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present work the feasibility of ␤-cyclodextrin in complexation was explored, as a tool for improving
the solubility and biological ability of daidzein derivatives. A series of phosphorylated daidzein deriva-
tives featuring different chain lengths were synthesized through a modified Atherton–Todd reaction and
their inclusion complexes with ␤CD were prepared by coprecipitation method. The inclusion complex-
ation behavior was studied by fluorescence, UV, FT-IR, MS and 1H NMR. The results showed that only
phosphorylated daidzein derivative carrying small substituent group ((C2H5O)2P O) entered the cavity
Received 11 August 2011
Received in revised form
29 September 2011
Accepted 6 October 2011
Keywords:
Phosphorylated daidzein
␤-Cyclodextrin
of ␤CD and formed 1:1 inclusion complex. The formation constant was 175 (mol/L)−1
.
Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Inclusion complex
1. Introduction
Introducing of phosphate ester to daidzein could change its physic-
ochemical properties and increase the biological ability through the
formation of non-covalent complexes with the proteins, while the
change of the solubility is quite limited [15,16]. To increase the sol-
ubility and biological ability of daidzein, we synthesized a series
of phosphorylated daidzein derivatives and studied their inclusion
with ␤CD using fluorescence, IR, MS and NMR techniques.
Cyclodextrins (CDs) are truncated, cone-shaped cyclic oligosac-
charides, composed of 6 (in ␣-), 7 (in ␤-), 8 (in ␥-) or more ␣-1,
4 linked glucoses and the most abundant natural cyclodextrin
is ␤-cyclodextrin (␤CD). Due to its special molecular struc-
ture/hydrophobic internal cavity and hydrophilic external surface,
␤CD can form inclusion complexes with various guest molecules
featuring suitable polarity and dimension [1]. Moreover, forma-
tion of ␤CD inclusion complex generates aqueous drug solutions
without the use of organic co-solvents, surfactants, or lipids, as
formulation adjuncts [2]. Recently, ␤CD inclusion complexes have
been used in pharmaceutical industry to increase the solubility, sta-
bility and bioavailability of the guest molecules [3]. Many poorly
soluble drugs have been converted to ␤CD inclusions and their
physicochemical properties could be significantly improved [4,5].
Due to the impressive biological activities of flavonoids,
they have attracted extensive interests, while research on the
interaction between flavonoids and ␤CD are quite rare [6,7].
Daidzein (7,4ꢀ-dihydroxyisoflavone) is one of the most impor-
tant isoflavonoids, which has been pharmacologically shown
featuring the bioactivities of anti-oxidant [8], anti-allergic, anti-
inflammatory [9], anti-microbial [10], anti-cancer [11–13], and
playing important role in female hormone [14]. However, the
bioavailability of daidzein by oral absorption is quite low
because of its poor solubility, which limits its clinical application.
2. Experimental
2.1. Materials
␤CD (98%) was purchased from Kermel (China) and recrys-
tallized from doubly distilled water before using. Daidzein was
purchased from Shanxi Huike Botanical Development Co. (China).
Dialkyl phosphates were synthesized in our lab. The water used was
doubly distilled, deionized and filtered through 0.22 ␮m Millipore
filters. All other materials were of analytical reagent grade purity.
2.2. Apparatus
UV–vis absorption spectra were recorded on a Spekol 2000
UV–vis double beam spectrophotometer using 1 cm quartz cell,
with a wavelength ranging between 200 and 400 nm. The fluo-
rescence spectra were recorded on a Cary Eclipse fluorescence
spectrometer using 1 cm quartz cell, 5 nm slit width, excitation at
257 nm and fluorescence emission obtained at 417 nm. Each mea-
surement was carried out in triplicate at 23 ◦C.
∗
IR spectra were recorded using an IR-200 Fourier transform
Corresponding author. Tel.: +86 371 67756712; fax: +86 371 67756718.
E-mail address: henangongda@yahoo.com (Y. Xiao).
spectrometer in KBr pellets. 1H NMR experiments were carried out
1386-1425/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.10.014

Y. Xiao et al. / Spectrochimica Acta Part A 85 (2012) 298–302
299
Scheme 1. Synthesis of phosphorylated daidzein derivatives.
using a Bruker Avance 400 MHz instrument in DMSO-d6-D2O (1:1,
v/v) mixture, using TMS as internal standard. Mass spectra were
analyzed on a Bruker Esquire 3000 mass spectrometer fitted with
an ion spray source working in positive ion mode, using methanol
as solvent.
compounds 3a–3g in distilled water and ␤CD in ethanol were com-
bined and stirred at 25 ◦C for 1 h. The mixture was concentrated
and cooled to 4 ◦C for 24 h. Collection of the precipitate gave the
inclusion complexes.
Sonication was performed using a Jiangsu Kunshan KQ-200VDE
ultrasonic cleaner.
2.4.3. Preparation of physical mixtures
Molar equivalent compounds 3a–3g and ␤CD were combined
and ground in an agate mortar until the mixture was homogeneous.
2.3. Procedure
3. Results and discussion
2.3.1. Phase-solubility measures
Phase-solubility measurements were carried out according to
the method of Higuchi and Connors [17]. A fixed amount of phos-
phorylated daidzein derivative, exceeding its solubility, was added
to 10 mL aqueous solutions of ␤CD with concentrations of 0,
1.0 × 10−3, 2.0 × 10−3, 2.5 × 10−3, 3.5 × 10−3, 4.0 × 10−3 mol/L in
capped tubes. The mixture was sonicated in ultrasonic cleaner for
20 min and then magnetically stirred for 3 days to reach equilib-
rium in a thermostated bath at 23 ◦C in darkness. The suspension
was filtered through Sartorius Minisart®-SRP 15 PTFE 0.45 ␮m fil-
ters, respectively. 1 mL filtrate was withdrawn from each vial and
then measured by UV–vis absorption spectroscopy (260 nm). Each
experiment was carried out in triplicate.
3.1. Fluorescence study
As a highly sensitive and selective technology, fluorescence
method has been extensively used to study the interaction between
host and guest molecules [18,19]. We synthesized a series of
phosphorylated daidzein derivatives 3a–3g through a modified
Atherton–Todd reaction, and studied their interaction with ␤CD
using fluorescence method (Figs. 1 and 2).
Fig. 1 showed that the fluorescence intensity of complex 3a
enhanced with concentration increasing of ␤CD. These data sug-
gested that a stable inclusion complex was formed between ␤CD
and compound 3a. Since the CD cavity provided an apolar environ-
ment for compound 3a and confined the motion of the molecule,
protecting the excited states of 3a molecule from nonradiative and
quenching processes that normally readily occurred in bulk aque-
ous solution [20], the quantum yield of the excited fluorophore of
compound 3a was increased in combination with ␤CD [21,22].
Fig. 2 showed that the fluorescence intensity of complex 3b
decreased with concentration increasing of ␤CD. The phenomena
maybe explained that the increased bulkiness of the phosphate
group in compound 3b made it difficult to enter the cavity of ␤CD
to form inclusion complex. Instead, there may exist the formation
2.3.2. Fluorescence spectra measurements
In the fluorescence spectra measurements of compound 3a,
different volumes of ␤CD (0, 1, 1.5, 2.5, 3, 4 mL) with concen-
tration of 0.01 mol/L was added to 1 mL compound 3a solution
(1 × 10−3 mol/L) and diluted to 10 mL with distilled water. In the
fluorescence spectra measurements of compound 3b, different vol-
umes of ␤CD (0, 1, 2, 2.5, 3, 4, 5 mL) with concentration of 0.01 mol/L
was added to 1 mL compound 3b solution (1 × 10−3 mol/L) and
diluted to 10 mL with distilled water.
2.4. Preparation of samples
2.4.1. Synthesis of phosphorylated daidzein derivatives 3a–3g
Synthesis of phosphorylated daidzein derivatives was shown in
Scheme 1. Daidzein (0.6 g, 2.36 mmol) was added to a solution of
NEt3 (0.8 mL) in DMF (6 mL) in a three-neck flask and the mixture
was stirred until all the daidzein was dissolved. The flask was put
into an ice-water bath and then a solution of dialkyl phosphate
(2.36 mmol) in CCl4 (6 mL) was added dropwise with vigorous
stirring. After addition, the ice-water bath was removed and the
reaction proceeded at room temperature for 24 h. The mixture was
filtered and the filtrate was evaporated in vacuo. The residue was
then dispersed in 30 mL water, filtered and further purified by col-
umn chromatography using chloroform–methanol (30:1, v/v) as
eluent.
2.4.2. Preparation of solid inclusion complexes
The inclusion complexes of compounds 3a–3g with ␤CD
were prepared by the coprecipitation method. Molar equivalent
Fig. 1. The fluorescence spectra of compound 3a combined with different concen-
trations of ␤CD, 0, 1 × 10−3, 1.5 × 10−3, 2.5 × 10−3, 3 × 10−3, 4 × 10−3 mol/L (from
bottom to top).

300
Y. Xiao et al. / Spectrochimica Acta Part A 85 (2012) 298–302
Fig. 4. Phase-solubility diagrams of compound 3a in aqueous solutions of ␤CD.
Fig. 2. The fluorescence spectra of compound 3b combined with different concen-
trations of ␤CD, 0, 1 × 10−3, 2 × 10−3, 2.5 × 10−3, 3 × 10−3, 4 × 10−3, 5 × 10−3 mol/L
(from top to bottom).
Fig. 4 showed the phase solubility diagrams of inclusion complex
3a-␤CD. It could be observed that the solubility of compound 3a
increased linearly as the concentrations of ␤CD increased. Accord-
ing to the Higuchi and Connors classification, the diagrams obtained
were of AL type, where the stoichiometry of the inclusion complex
was assumed to be 1:1, in consistent with the result from fluores-
cence study. Compound 3a had a solubility of 1.19 × 10−3 mol/L
in water, while the solubility of 3a in a 1:1 inclusion complex
was increased to 2.2 × 10−3 mol/L. This phenomenon could be
attributed to the interaction between host/guest molecules, during
the supramolecular complex formation.
of complexes between ␤CD, solvent molecules and compound 3b,
which led to the enhanced quenching processes with the increas-
ing of ␤CD. The fluorescence spectra of complexes 3c–3g combined
with different concentrations of ␤CD showed the same trend with
those of complex 3b.
The formation constant can be obtained from fluorescence data
in Fig. 1 by the modified Benesi–Hildebrand equation [23,24].
1
1
1
=
+
F
ꢀ
ꢁ
n
F − F0
∞ − F0
3.3. FT-IR spectra studies
K F∞ − F
␤CD
[
]
0
Here F is the observed fluorescence intensity of compound
3a solution at each ␤CD concentration; F0 presents fluorescence
intensity of compound 3a solution in the absence of ␤CD. K is for-
mation constant of the complex; n represents the stoichiometry of
the complex formed. The plot curve of 1/(F − F0) against 1/[␤CD]2
deviated from linearity (data not shown), while the plot curve
of 1/(F − F0) against 1/[␤CD] (Fig. 3) exhibited excellent linearity,
which indicated that compound 3a formed a 1:1 stoichiometry
inclusion complex with ␤CD. The calculated formation constant K
The FT-IR spectroscopic analysis confirmed the interaction and
inclusion complex formation between compound 3a and ␤CD
[27,28]. Fig. 5 showed the FT-IR spectra of ␤CD (a), compound 3a (b),
3a/␤CDphysicalmixture(c) andinclusioncomplex(d). Comparison
of these spectra indicated the significant change in the charac-
teristic bands of the pure substances. In the inclusion complex,
absorbances at 1650–1700 cm−1 and 1250 cm−1, characteristic
bands attributed to the carbonyl and P O groups, disappeared
respectively. Meanwhile, the characteristic bands of daidzein at
was 1.75 × 102 (mol/L)−1
.
3.2. Solubility studies
It has been extensively reported in the literatures that ␤CD
molecules are able to increase the guest molecule solubility [25,26].
Fig. 5. FT-IR spectra of (a) ␤CD, (b) compound 3a, (c) physical mixture of 3a and
␤CD and (d) inclusion complex 3a-␤CD.
Fig. 3. Benesi–Hildebrand plots for 1:1 inclusion of compound 3a with ␤CD.

Y. Xiao et al. / Spectrochimica Acta Part A 85 (2012) 298–302
301
Fig. 6. Mass spectrum of 3a-␤CD.
1480–1600 cm−1 also disappeared in the FT-IR spectrum of the
inclusion complex. Additionally, in the inclusion complex, the
intensity and shape of these bands changed obviously compared
to those of compound 3a and physical mixture. The dramatic dif-
ferences indicated that the bending and vibrating of the structure
of compound 3a were restricted, resulting from the interaction of
compound 3a with the cavity of ␤CD.
the aromatic protons of the A- and C-rings, indicating that A- and
C-rings probably entered the inner cavity of ␤CD.
4. Conclusions
In this study, we synthesized a series of phosphorylated
daidzein derivatives through a modified Atherton–Todd reaction,
and studied their interaction with ␤CD. The data obtained from
fluorescence, UV–vis, FT-IR, MS and NMR studies showed that phos-
phorylated daidzein derivatives carrying small substituent (3a)
entered the cavity of ␤CD and formed 1:1 inclusion complex with
the formation constant being 175 (mol/L)−1, while phosphorylated
daidzein derivatives carrying bulky substituent (3b–3g) failed to
form inclusion complexes with ␤CD.
3.4. MS studies
Mass method is already known as a powerful tool to detect the
formation of complex. Fig. 6 showed the mass spectrum of the
inclusion complex 3a-␤CD. Ion peaks at m/z 1543.3 and 390.9 cor-
responded to [3a-␤CD+H2O+H]+ and compound 3a, respectively.
Acknowledgements
3.5. 1H NMR studies
We thank the Science and Technology hall of Henan Province
(Nos. 072300420070 and 82102330014), China, for financial sup-
port to this work.
Further support for the inclusion complex formation can be
obtained using 1H NMR spectroscopy, which has proved to be use-
ful in the study of ␤CD inclusion complex [29]. Here the formation
of inclusion complex could be proved from the comparison of the
chemical shifts of 3a before and after interaction with ␤CD, which
was defined as ꢀı. The broadening of the proton signals of 3a in
the presence of ␤CD suggested that 3a entered the cavity of ␤CD
and the motions of these protons were restricted [30]. Table 1 listed
the characteristic chemical shifts of 3a, 3a-␤CD and ꢀı in 1H NMR
spectra. Downfield shifts were observed for H-2, H-6 and H-8 of
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.10.014.
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