Studying on inclusion complexes of Wogonin with β-cyclodextrin and hydroxypropyl-cyclodextrin

Article abstract of DOI:10.1016/j.saa.2011.10.057

The formation of the complexes of Wogonin with β-cyclodextrin (β-CD) and hydroxypropyl-cyclodextrin (HP-β-CD) was studied by fluorescence spectra and nuclear magnetic resonance spectroscopy (NMR). The formation constants (Ks) of complexes were determined

Full text of DOI:10.1016/j.saa.2011.10.057

Spectrochimica Acta Part A 87 (2012) 25–28
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Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Studying on inclusion complexes of Wogonin with ␤-cyclodextrin and
hydroxypropyl-cyclodextrin
Jinxia Lia,∗, Jianbin Chaob,∗∗, Min Zhangb
a The Medical School of Shanxi Datong University, Datong 037009, PR China
b College of Chemistry and Chemical Engineering of Shanxi University, Taiyuan 030006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2011
Received in revised form 16 October 2011
Accepted 28 October 2011
The formation of the complexes of Wogonin with ␤-cyclodextrin (␤-CD) and hydroxypropyl-cyclodextrin
(HP-␤-CD) was studied by fluorescence spectra and nuclear magnetic resonance spectroscopy (NMR). The
formation constants (Ks) of complexes were determined by fluorescence method. The results suggested
that HP-␤-CD was easier to form inclusion with Wogonin than ␤-CD in solution. In different pH solutions,
CDs have different inclusive capacity to Wo. ␤-CD was most suitable for inclusion of neutral form and
HP-␤-CD was suitable for acidic form. In addition, the experimental resulted confirmed the existence of
1:1 inclusion complex of Wogonin with CDs.
Keywords:
Wogonin
Cyclodextrins
Fluorescence
NMR
Besides, kinetic studies of DPPH• with Wogonin and CDs complexes were done. The results obtained
indicated that the complex was the most reactive form. Special configuration of complex has been
proposed on NMR technique.
DPPH•
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
when these compounds from inclusion complexes [10]. High res-
olution nuclear magnetic resonance (NMR) is a powerful tool for
Investigations of molecular recognition have attracted much
attention in supramolecular chemistry involving natural and arti-
ficial host–guest systems [1]. Cyclodextrin complex has been
successfully used to improve the solubility, chemical stability and
bioavailability of a number of poorly soluble compounds [2–4].
Recently, various hydrophilic, hydrophobic and ionic cyclodextrin
derivatives have been utilized to extend the physicochemical prop-
erties and inclusion capacity of natural cyclodextrin [5,6]. HP-␤-CD
is a water-soluble derivative of ␤-CD, which has been widely stud-
ied as a complexion agent for many pharmaceuticals. The ability of
CDs to form inclusion complexes is highly affected by size, shape,
hydrophobicity and the form of the guest’s molecular.
Wogonin (Wo, Fig. 1), a flavonoid present in the root of
Scutellaria baicalensis Georgi, has attracted considerable attention
because of the activities, such as anti-respiratory syncytial virus
[7], anti-tumor effects [8] and anti-hepatitis B virus [9]. However,
in spite of the wide spectrum of pharmacological properties, its use
in pharmaceutical field is limited because of its poor solubility.
Since many guest compounds present fluorescent properties, it
is interesting to analyze the changes produced in such properties
studying CD complexes [11].
The inclusion of Wogonin with biologic-molecular for example
human gammaglobulin [12], human serum albumin [13] and have
DNA [14] been researched by many groups, but the inclusion of
Wogonin with cyclodextrins has not been investigated. Such, this
paper has definite guidance purport in clinic pharmaceutics. In this
paper, the interaction of Wo with ␤-CD and HP-␤-CD were studied
in detail based on Fluorescence method and NMR, and determined
the effect of the complexation process on their antioxidant capacity.
A new theories basis on new drug-carriers system was supplied.
2. Experimental
2.1. Apparatus and materials
Fluorescence measurements were performed by F-2500 FL spec-
trofluoremeter (Hitachi) using 1 cm quartz cell and both the slits
were set at 20 nm with the excitation wavelength at 270 nm and
the emission at 360 nm. All the NMR date was obtained on Bruker
Avance DRX 300 MHZ NMR spectrometer.
The stock solution of 1.0 × 10−4 mol/L Wo (provided by Dr.
Zhang and was purified by recrystallization) was prepared by
dissolving and diluting its crystals in water. 2 ml of 0.2 mol/L phos-
phate buffer solution was used to control the pH value of the
media. CDs and DPPH• were purchased from Sigma–Aldrich, Inc., St.
Louis, MO. All other reagents were of analytical-reagent grade and
∗
Corresponding author. Tel.: +86 13935238620; fax: +86 3526033101.
Co-Corresponding author at: The Institute of Applied Chemistry of Shanxi Uni-
∗∗
versity, Taiyuan 030006, People’s Republic of China.
E-mail address: ljx-2000@tom.com (J. Li).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.10.057

26
J. Li et al. / Spectrochimica Acta Part A 87 (2012) 25–28
Fig. 1. The chemical structure of wogonin.
were used without purification. Doubly distilled water was used
throughout. All experiments were carried out at 20 1 ◦C.
2.2. Experimental Procedure
A 0.1 or 1 ml aliquot of the stock solution (1.0 × 10−4 mol/L) of
Wo was transferred into a 10 ml volumetric burette, and an appro-
priate amount of 1.0 × 10−2 mol/L CDs (␤-CD and HP-␤-CD) was
added. The solution was diluted to a final volume of 10 ml with
distilled water. The mixed solution was diluted to the final volume
with distilled water and ultrasonic handled for 30 min, equilibrated
for 30 min at 20 1 ◦C. The working solution was transferred into a
1 cm × 1 cm quartz cell to record fluorescence spectra. All measure-
ments of fluorescence were made against a blank solution treated
in the same way but without Wo in a 1.0 cm quartz cell.
2.3. NMR measurements
Fig. 2. Fluorescence emission spectra of 1.0 × 10−6 mol/L Wogonin in CDs. (A):
␤-CD; (B): HP-␤-CD. CD concentration (M): ␤-CD: 0–6.0 × 10−3; and HP-␤-CD:
All the concentrations of Wo and CDs solution were
1.0 × 10−4 mol/L and Wo solution is diluted with CDs solutions,
respectively, at the volume ratio of 1:1. 1H NMR of Wo solution as
well as its inclusion complexes solutions was also performed to get
further evidence. D2O was as solvent. 1H NMR spectra was obtained
at 300.13 MHz with 10 ␮s as 90◦ pulse width. All experiments were
performed at 20 1 ◦C.
0–7.0 × 10−3
.
3. Results and discussion
3.1. Fluorescence study
Fig. 2 shows fluorescence spectra of Wogonin in the absence and
presence of CDs (including ␤-CD, and HP-␤-CD). The maximum
excitation and emission wavelengths were 270 nm and 362 nm,
respectively. With increasing concentration of CDs, the emission
peaks appeared blue shift, and a new increasing emission wave-
length was observed with the concentration increasing of HP-␤-CD
at about 440 nm. These suggested that the inclusion complexes
were likely formed between Wo and CDs. The CDs cavities provided
an apolar environment for the Wo molecular, and thus increase the
fluorescence of Wo.
2.4. Determination of antioxidant activity by the scavenging of
the stable radical DPPH•
The antioxidant activity was measured, wherein the bleaching
rate of a stable free radical, DPPH• is monitored at a characteristic
wavelength in the presence of the sample. In its radical form, DPPH•
absorbs at 517–520 nm, but upon reduction by an antioxidant or a
radical species its absorption decreases.
A volume of 2 ml of 1.0 × 10−5 M DPPH• was used. Furthermore,
DPPH• is insoluble in aqueous solution the scavenging study was
performed in mixture of ethanol–water (20:80).
The reaction was started by addition of 1 ml of Wo
(1.0 × 10−5 M), Wo/␤-CD, and Wo/HP-␤-CD complex samples,
which correspond to the 3 mM cyclodextrin concentration. The
bleaching of DPPH• was followed at 520 nm.
The decrease in absorbance at 520 nm was measured against a
blank of ethanol–water (20:80) 1 ml and 2 ml 1.0 × 10−5 M DPPH• to
estimate the radical scavenging capacity of each antioxidant sam-
ple. The results were expressed as percentage DPPH· elimination
calculated according to the following equation [15]:
The inclusion formation constant (K) is a measure of the com-
plexing power of CD. The formation constant and ratio of the
complex were obtained from fluorescence data using the modified
Benesi–Hildebrand equation [16]
1
1
1
˛
=
+
(2)
F − F0
[CDs]K˛
where, F and F0 represent the fluorescence intensity of Wogonin
in the presence and absence of CDs, respectively; K is a forming
constant; ˛ is a constant.
Fig. 3 shows the double reciprocal plots of 1/(F − F0) versus
1/[CD]. They exhibit good linearity. These implied that the inclu-
sion complexes have a stoichiometry of 1:1. The binding constant
(Ks) of the complexes at different pH (including pH3.50, pH6.50,
pH7.50 and pH10.50) were shown in Table 1. As shown in Table 1,
the binding constant and solubility of Wo determined with CDs
followed the rank order HP-␤-CD > ␤-CD. HP-␤-CD was suitable for
1 − As
AU =
× 100,
(1)
A0
where AU is radical-scavenging activity, As is absorbance of sample
and A0 absorbance of blank sample.

J. Li et al. / Spectrochimica Acta Part A 87 (2012) 25–28
27
Fig. 3. The double reciprocal plots for Wogonin complexes to ␤-CD or HP-␤-CD at
pH 6.50. (ꢀ): ␤-CD; and (ꢁ): HP-␤-CD.
Table 1
Apparent stability constant (Ks) of Wo inclusion at different pH, the temperature is
at 20 1 ◦C.
pH
Wo/HP-␤-CD
Wo/␤-CD
Fig. 4. 1H NMR spectra of Wo and inclusion complexes: the order were Wo, Wo/␤-
CD and Wo/HP-␤-CD from the below to the up.
K
r2
K
r2
3.50
6.50
7.50
370
0.9991
0.9719
0.9834
0.9929
45
133
60
67
0.9814
0.9990
0.9904
0.9855
220
138
72
10.50
Table 2
1H NMR chemical shifts corresponding to Wo in the absence and presence of CDs in
D2O.
Wo (H)
Wo (ı0)
Wo/␤-CD (ı1)
ꢀı1
WO/HP-␤-CD(ı2)
ꢀı2
H-6
H-3
6.313
7.002
7.629
8.085
6.312
6.999
7.628
8.066
0.001
0.003
0.001
0.019
6.311
6.990
7.626
8.063
0.002
0.010
0.003
0.021
H-3ꢀ4ꢀ5ꢀ
H-2ꢀ6ꢀ
the inclusion with Wo in acidic media, and ␤-CD was suitable for
the inclusion in neutral media. That was associated to the molecule
dissociation in different pH values.
3.2. NMR
Fig. 5. 1H NMR spectra of ␤-CD and Wo/␤-CD inclusion complex from the below to
the up.
To ascertain the structure of the inclusion complexes between
Wo and CDs, 1H-NMR spectroscopy studies of free drug and inclu-
sion complexes were therefore undertaken. Figs. 4 and 5 illustrated
the change of hydrogen atom of Wo and CDs before and after form-
ing the inclusion complexes. The difference in hydrogen chemical
shift values between Wo in the free and complexed state were pre-
sented in Table 2. It can be seen from the figures that the H-3 and
H-2ꢀ, H-6ꢀ of Wo exhibited larger chemical shifts change after form-
ing inclusion with ␤-CD, namely, the H-3 and H-2ꢀ, H-6ꢀ of Wo were
all entered into the cavity of ␤-CD, while H-6 and H-3ꢀ, 4ꢀ 5ꢀ were
not into the ␤-CD cavity. But in the inclusion with HP-␤-CD, H-6,
H-3 and H-2ꢀ, H-6ꢀ and H-3ꢀ, 4ꢀ, 5ꢀ of Wo exhibited larger chemical
shifts change, which indicated that the A–C rings of Wo were all
entered into the cavity of HP-␤-CD.
chemical shift than H-5, which illustrated that the molecular of Wo
entered into the cavity of ␤-CD from the large port; while The H-5
of HP-␤-CD had larger chemical shift than H-3, which illustrated
that the molecular of Wo entered into the cavity of HP-␤-CD from
the ventage port.
From all the above, the mechanism of complex between Wo and
CDs were shown as follow (Fig. 6).
3.3. Scavenging study of DPPH• by free or complexed-Wo
DPPH• is a stable free radical generating a deep violet solution in
organic solvents. Its progressive discoloration when in the presence
of Wo indicated that it is acting as an antioxidant.
Table 3 showed the hydrogen chemical shift change values of
CDs after forming the complexes. The H-3 of ␤-CD had larger
Table 3
1H NMR chemical shifts CDs and the inclusion complexes in D2O.
CD (H)
␤-CD (ı0)
Wo/␤-CD (ı1)
ꢀı1
0.058
0.034
0.096
0.064
−0.128
Wo-␤-CD (ı0ꢀ)
Wo/HP-␤-CD (ı2)
ꢀ␦2
H-4
H-2
H-5
H-6
H-3
3.376
3.413
3.633
3.696
3.728
3.318
3.379
3.547
3.632
3.856
3.260
3.346
3.461
3.615
3.749
3.300
3.459
3.614
3.762
3.858
−0.040
−0.113
−0.153
−0.147
−0.109

28
J. Li et al. / Spectrochimica Acta Part A 87 (2012) 25–28
factors of affecting guest/host binding are size matching between
CD and guest, and the hydrophobicity of the guest molecule. Among
the CDs examined, HP-␤-CD was the most suitable for the inclu-
sion of Wogonin in solution, ␤-CD was most suitable for inclusion
of neutral form and HP-␤-CD was suitable for acidic form. This
suggested that HP-␤-CD cavity supplied more hydrophobic envi-
ronment. And the activity of eliminating free radical DPPH• were
inclusion complexes > free Wogonin. In addition, the fluorescence
spectroscopy and 1H NMR data have shown the formation of a sta-
ble 1:1 stoichiometri complex of Wogonin with ␤-CD and HP-␤-CD.
Moreover, the present study demonstrated that CDs served as drugs
carrier system in a dosage-controlled manner and can increase the
solubility, stability and antioxidant activity of guest molecular. A
mechanism was set up to expound the structure of the inclusion
complexes.
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Furthermore, since the mechanism of DPPH• reduction is
known, the amount remaining of both reagents may be determined.
The rate of the DPPH•-scavenging reaction was measured by
monitoring the decrease in absorbance at 520 nm due to DPPH•.
The conclusion showed the consumption of DPPH• which indicates
that the complexed Wo/CDs were more effective than free Wo, with
the HP-␤-CD complex (44.44%) = ␤-CD complex (44.44%) > free Wo
(22.22%). The scavenging ability was measured as a relative scav-
enging in presence of free or complex Wo. That was in according
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4. Conclusion
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The present study has demonstrated the inclusion complex
interaction between Wogonin with ␤-CD and HP-␤-CD. The major