Thermodynamic study on the effects of β-cyclodextrin inclusion with berberine

Article abstract of DOI:10.1016/S1386-1425(01)00536-4

The fluorescence enhancement of berberine (Berb) as a result of complex with β-cyclodextrin (β-CD) is investigated. The association constants of α-CD and β-CD with Berb are 60 and 137 M^-1 at 20°C in pH 7.20 aqueous solution. Effects of temperature on the forming inclusion complexes of β-CD with Berb have been examined through using fluorescence titration. Enthalpy and entropy values calculated from fluorescence data are -33.7 kJ·mol^-1 and 74.3 J·mol^-1·K^-1, respectively. It was found that the dielectric constant of β-CD cavity is about 24 in a rough analogy with absolute alcohol. These results suggest that the extrusion of 'high energy water' molecules from the cavity of β-CD and hydrophobic interaction upon the inclusion complex formation are the main forces of the inclusion reaction. Effect of pH on the association of β-CD with Berb was also studied. Mechanism of the inclusion of β-CD with Berb is further studied by absorption and NMR measurements. Results show that β-CD forms a 1:1 inclusion complex with Berb.

Full text of DOI:10.1016/S1386-1425(01)00536-4

Spectrochimica Acta Part A 58 (2002) 249–256
www.elsevier.com/locate/saa
Thermodynamic study on the effects of b-cyclodextrin
inclusion with berberine
Jun-Sheng Yu *, Fang-Di Wei, Wei Gao, Chang-Chun Zhao
Department of Chemistry, Institute of Analytical Science, Nanjing Uni6ersity, Nanjing 210093, People’s Republic of China
Received 18 December 2000; accepted 21 April 2001
Abstract
The fluorescence enhancement of berberine (Berb) as a result of complex with b-cyclodextrin (b-CD) is investigated.
−
1
The association constants of a-CD and b-CD with Berb are 60 and 137 M
at 20 °C in pH 7.20 aqueous solution.
Effects of temperature on the forming inclusion complexes of b-CD with Berb have been examined through using
fluorescence titration. Enthalpy and entropy values calculated from fluorescence data are −33.7 kJ mol
.
−1
and 74.3
−
1
−1
J·mol ·K , respectively. It was found that the dielectric constant of b-CD cavity is about 24 in a rough analogy
with absolute alcohol. These results suggest that the extrusion of ‘high energy water’ molecules from the cavity of
b-CD and hydrophobic interaction upon the inclusion complex formation are the main forces of the inclusion
reaction. Effect of pH on the association of b-CD with Berb was also studied. Mechanism of the inclusion of b-CD
with Berb is further studied by absorption and NMR measurements. Results show that b-CD forms a 1:1 inclusion
complex with Berb. © 2002 Elsevier Science B.V. All rights reserved.
Keywords: Berberine; Cyclodextrin; Inclusion complex
driving forces have been proposed for the inclu-
1. Introduction
sion of CDs with substrates [4–6]: hydrogen bind-
ing, Van der Waals force, hydrophobic
interaction and the release of ‘high energy water’
molecules from the cavity. As a result of complex
formation, the characteristic properties of the in-
cluded substance, such as solubility [7], chemical
reactivity [8] and spectral properties [9] will be
changed. Thus, CDs have been applied to several
areas of science and technology [10]. In the phar-
maceutical industry, CDs have been used to en-
hance the solubility, stability and bioavailability
of drugs [11–13]. b-CD is the favorite encapsula-
tion of drugs, for its lower price and higher
productive rate.
Cyclodextrins (CDs) are toroidally shaped
polysaccharides made up of six to eight D-glucose
monomers connected at 1 and 4 carbon atoms [1].
The cavity of CDs is relatively hydrophobic com-
pared to water, while the external faces are hy-
drophilic [2]. They are capable of discerning
various types of guest molecules by selectively
incorporating such molecules through size and
polarity consideration [3]. Up to now, several
*
Corresponding author. Fax: +86-25-3317761.
E-mail address: ndchemyu@public1.ptt.js.cn (J.-S. Yu).
1
386-1425/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S1386-1425(01)00536-4

2
50
J.-S. Yu et al. / Spectrochimica Acta Part A 58 (2002) 249–256
Berberine (Berb) is the main effective content
Shimadzu UV-3100 spectrophotometer. NMR
spectra were collected on a Bruker-300 MHZ
nuclear magnetic resonance spectrometer. Sam-
ple temperature for the NMR experiments was
maintained at 22.090.1 °C.
in many Chinese drugs, such as coptis and phel-
lodendron. Berb has an effective function of an-
tibiosis, chloleretica and antihypertension, but it
is poorly absorbed by the body [14,15]. It is
very important to study the mechanism of inclu-
sion of Berb with b-CD, for it may enhance the
absorption of Berb and can provide better un-
derstanding of drug complex kinetics and bio-
logical transport properties.
It is the aim of this work: (a) to show that
b-CD can indeed form inclusion compounds
with Berb; (b) to study the thermodynamic
properties of the b-CD and Berb system; (c) to
characterize the mechanism of the complex for-
mation. We use steady-state fluorescence spec-
troscopy to study the inclusion of Berb with
b-CD, results are shown for the determination
of various thermodynamic parameters (equi-
librium constants, enthalpy and entropy) and ef-
fects of pH on the inclusion reaction. The
equilibrium constant of the inclusion of Berb
with a-CD at 20 °C are also given. It is resulted
that the size of CDs has an important effect on
the inclusion. Experiment results of Berb in var-
ious dielectric microenvironment indicate that
the dielectrics of b-CD cavity is analogous to
that of absolute alcohol (m:24). In addition,
absorption and NMR measurements are used to
further study the mechanism of the inclusion re-
action.
2.2. Materials
The Berb hydrochloride (97%), a-CD (98%)
and b-CD (99%) were all purchased from Acros.
The NMR samples were prepared with deuter-
ated water (99.8% atom D) purchased from Bei-
jing Chemical Factory. Reagents for buffered
aqueous solutions and solvent were all analytical
reagent grade. All chemicals were used without
further purification.
2.3. Method
2.3.1. Fluorescence studies
Sample concentration of a-CD, b-CD ranged
−
3
from zero up to 3.00×10
M was used. Berb
hydrochloride concentration in each sample was
−
5
always 2.00×10 M. For the pH and solvent
studies, a constant temperature of 10 °C was
used. In the case of temperature studies, temper-
ature of 5, 10, 15, 20, 25, 30, 40 °C was used.
Except for the pH studies, a phosphate buffered
aqueous solution of pH 7.20 was used and the
−
1
concentration of it remained at 13 mmol l
.
All samples were prepared with double distilled
water.
2. Experimental section
2.3.2. Absorption studies
The b-CD concentration ranged from zero to
2
.1. Apparatus
−
3
1
.18×10 M and the Berb hydrochloride con-
−
5
centration was maintained at 2.00×10
M.
All steady-state fluorescence measurements
The blanks were prepared with appropriate con-
centration of b-CD, phosphate buffered aqueous
solution (pH 7.20) and double distilled water for
absorption measurements.
were made with a Perkin-Elmer LS-50B fluores-
cence spectrometer equipped with an Epson 386
computer, a thermostated cell holder with auto-
matic stirrer and temperature readings (Perkin-
Elmer accessory, No. L2250141). Fluorescence
emission spectra were acquired with an excita-
tion wavelength at 348 nm and the emission in-
tensities were monitored at 530 nm. Excitation
and emission bandwidths were set at 5 nm. Ab-
sorption measurements were carried out on a
2.3.3. NMR studies
Proton NMR spectra were recorded in D O
2
at ambient temperature. Chemicals shifts are ex-
pressed in parts per million (ppm) relative to
HOD at 4.7 ppm.

J.-S. Yu et al. / Spectrochimica Acta Part A 58 (2002) 249–256
251
3. Results and discussion
3.1. Fluorescence measurement
Fig. 1 shows fluorescence spectra of Berb in
phosphate buffered aqueous solution (pH 7.20)
containing b-CD at various concentrations. The
fluorescence intensity of Berb at 530 nm is
markedly enhanced with the increasing of b-CD
concentration. The fluorescence intensity at 460
nm remains unchanged. The fluorescence en-
hancement of Berb at 530 nm in the presence of
b-CD is more than 10 times that in the absence of
b-CD. The trend is analogous to the fluorescence
enhancement of benzene derivatives by forming
inclusion complexes with b-CD in aqueous solu-
tions, reported by Hoshino et al. [16]. Since the
emission band at 530 nm is attributed to the
isoquinoline moiety of Berb molecule, it may be
inferred that the isoquinoline moiety of the
molecule penetrates into the b-CD cavity. Fig. 2
shows fluorescence spectra of Berb in different
dielectric solvent. It can be seen that the fluores-
cence enhancement of Berb molecule in absolute
alcohol is almost the same as that of Berb form-
ing inclusion complex with b-CD in pH 7.20
aqueous solution (Fig. 1). This suggests that the
−
5
Fig. 2. Fluorescence spectra of berberine (2.00×10
M) in
different solvent: (a) water; (b) alcohol/water (v:v=1:1); (c)
acetonitrile; (d) absolute alcohol; (e) n-propanol.
fluorescence enhancement of Berb in the presence
of b-CD is due to the hydrophobic interaction of
b-CD cavity. Since the dielectric constant of abso-
lute alcohol is 24.3 at 25 °C, it may be estimated
that the dielectric constant of b-CD cavity is
approximately to 24 at 25 °C for Berb molecules.
3.2. Absorption measurement
Inclusion complex formation often causes
changes in absorbance spectra of substrates. The
fact that whether isosbestic point appears or not
and how many isosbestic points occur has been
used to determine the formation of b-CD/guest
inclusions [17,18]. Fig. 3 depicts the general de-
creasing trend in absorbance with increasing the
concentration of b-CD. The isosbestic point ap-
pears at 365nm, which indicates that the inclusion
complex of b-CD with Berb has been produced in
pH 7.20 aqueous solution. The spectra shows
clearly that addition of b-CD yields the hypo-
chromism and a small red shift. Hypochromism
was suggested to be due to the interaction be-
tween the electronic states of Berb molecule with
those of b-CD. The red shift also supports some
H-bonding interactions of Berb with b-CD.
−
5
Fig. 1. Fluorescence spectra of berberine (2.00×10
M) at
3
.3. Stoichiometric ratio and effect of CD size
various concentrations of b-CD: (a) without b-CD; (b) 2.00×
on binding strength between Berb and CD
−
4
−4
−4
1
0
M b-CD; (c) 5.00×10
M b-CD; (d) 8.00×10
M
−
3
−3
b-CD; (e) 1.20×10
M b-CD; (f) 1.60×10
M b-CD; (h) 2.60×10
M b-CD; (g)
M b-CD; (i) 3.00×
−
3
−3
Information concerning the exact stoichiometry
and arrangement of Berb within or in association
2
.00×10
−
3
1
0
M b-CD.

2
52
J.-S. Yu et al. / Spectrochimica Acta Part A 58 (2002) 249–256
Table 1
Equilibrium constant values for the Berb/a-, b-CD complex
20 °C, pH 7.20)
(
Berb/CD
K (M−1)
Berb/a-CD
Berb/b-CD
60
137
The association constant, K, for the inclusion
reaction
Berb+CDUBerb · CD
is given by
(1)
K=[Berb · CD]/[CD][Berb]
(2)
−
5
Fig. 3. Absorption spectra of berberine (2.00×10
M) at
various concentrations of b-CD: (a) without b-CD; (b) 3.23×
The mass-balanced expressions may be substi-
tuted such that
−
4
−4
−4
1
0
M b-CD; (c) 4.76×10
M b-CD; (d) 6.25×10
M
−
4
−3
b-CD; (e) 9.09×10
M b-CD; (f) 1.04×10
M b-CD; (g)
−
3
1
.18×10
M b-CD.
K=[Berb · CD]/(CCD−[Berb · CD])
(
CBerb−[Berb · CD])
(3)
with the CD molecule is crucial to the under-
standing of the mechanism of the inclusion of
Berb with CD. Benesi–Hildebrand plots have
been widely utilized to further characterize the
stoichiometry and strength of association of
CD/guest complex [19,20]. A Benesi–Hildebrand
plot at 20 °C assuming a 1:1 association be-
tween a-, b-CD and Berb reveals a linear regres-
sion with a correlation of 0.993 and 0.999,
respectively (Fig. 4).
where CCD and CBerb are the analytical concen-
tration of CD and Berb, respectively. Because
we always employ a large excess of CD relative
to Berb, we can assume that CCDꢀ[Berb · CD],
thus, Eq. (3) becomes
K=[Berb · CD]/CCD(CBerb−[Berb · CD])
(4)
The fluorescence intensity of the complex,
FBerb · CD, is given by
Benesi–Hildebrand equation is the underlying
theory for employing steady-state fluorescence
intensities to calculate equilibrium constants.
FBerb · CD=QBerb · CD[Berb · CD]ki
(5)
where QBerb · CD is the quantum efficiency of the
complex and ki is an instrumental constant.
Substitution of Eq. (5) into Eq. (4) generates the
useful Benesi–Hildebrand relation.
−
1
CBerb/FBerb · CD=(Kk Q
· CDCCD)
i
Berb
−
1
+
(k Q
i
· CD
)
(6)
Berb
Using
a
double-reciprocal plot of (CBerb
/
FBerb · CD) versus (1/CCD), the association con-
stant is determined from the ratio of the
intercept to the slope.
Table 1 shows the equilibrium constant values
for the Berb/a-, b-CD complexes. At the same
condition, the equilibrium constant for the Berb/
a-CD complex is smaller than that for the Berb/
Fig. 4. Benesi–Hidebrand plots for the (a) Berb/a-CD and (b)
Berb/b-CD complex.

J.-S. Yu et al. / Spectrochimica Acta Part A 58 (2002) 249–256
253
b-CD complex. This suggests that the size of CD
has an essential effect on the inclusion of CD with
Berb.
3.4. Effect of pH on the inclusion of i-CD with
Berb
Fig. 5 shows fluorescence intensity of Berb/b-
CD complex in different pH solutions containing
various concentration of b-CD (from zero to
−
3
−5
3
.00×10 M) and Berb (always 2.00×10 M).
The fluorescence intensity of Berb/b-CD system
versus b-CD concentration is a linear plot, and it
can be seen that the slope of the linear plot
increases gradually with the system pH from 1.20
to 7.20 (Fig. 5A), and decreases from pH 7.20 to
pH 11.20 (Fig. 5B). Fig. 5C shows clearly that the
tendency of fluorescence enhancement of Berb/b-
Fig. 6. Benesi–hildebrand plots for the Berb/b-CD complex at
different temperature: (a) 5 °C; (b) 10 °C; (c) 15 °C; (d)
2
0 °C; (e) 25 °C; (f) 30 °C; (g) 40 °C.
CD system is varied with pH in aqueous solution,
and fluorescence intensity of Berb/b-CD system
approaches to the maximum at pH 7.20. Appar-
ently, an acid or alkaloid environment tends to
retard the inclusion of b-CD with Berb. The
fluorescence intensity of Berb molecule is approxi-
mately constant in the range of pH 1.20–11.20. It
is probable that b-CD is stable in alkaline solu-
tions and susceptible to acid hydrolysis [2], and as
pH increases, the Berb becomes ionized to a
greater extent. This may tend to partially offset
the desire on the part of the hydrophobic portion
of Berb molecule to reside in b-CD cavity.
3.5. Effect of temperature on the inclusion of
i-CD with Berb
Fig. 6 is the Benesi–Hildebrand plot of Berb/b-
CD complex at seven different temperatures. The
slope and intercept are all increased with the
increasing of temperature. The equilibrium con-
stant for Berb/b-CD complex calculated from the
ratio of the intercept to the slope is given in Table
2
. It may be seen from Table 2 that the values of
Table 2
Equilibrium constant values at seven different temperatures
for the Berb/b-CD complex (pH 7.20)
Fig. 5. Fluorescence intensity of Berb/b-CD complex at vari-
ous pH values: (a) 1.20; (b) 2.00; (c) 3.00; (d) 4.00; (e) 5.20; (f)
T (°C)
5
337
10
197
15
151
20
137
25
87
30
77
40
64
K (M−1)
7
.20; (g) 9.20; (h) 10.20; (i) 11.20.

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54
J.-S. Yu et al. / Spectrochimica Acta Part A 58 (2002) 249–256
Fig. 7. 1H NMR spectra of b-CD and berberine in D O: (a) 1.00×10
−3
M b-CD; (b) 2.60×10
−4
M berberine; (c) 1.00×10−3
2
−
4
M b-CD with 2.60×10
M berberine. Spectra were acquired with a 300-MHz Nuclear Magnetic Resonance Spectrometer.
equilibrium constant of Berb/b-CD system de-
crease with the increasing of temperature. This is
resulted from the increase of collision between
molecules with the increasing of temperature. A
plot of −R ln K versus 1/T was linear with a
correlation coefficient of 0.972 (seven points). The
value of enthalpy calculated from the slope is
3.6. NMR study
The mechanism of interaction between Berb
and b-CD was investigated by using nuclear mag-
netic resonance (NMR) spectroscopy. The basis
of information gained from NMR spectroscopy is
located in the shifts, the loss of resolution and
broadening of signals observed on Berb/b-CD
protons upon addition of the guest/host molecule.
Fig. 7 shows the qualitative features concerning
the interaction of Berb with b-CD. The NMR
spectrum of b-CD (Fig. 7a) consists of peaks from
five kinds of protons [22,23]: the H-1 doublet (at
l 4.97), the H-3 triplet (at l 3.89), a strong
unresolved broad peak consisting of H-6 and H-5
(at l 3.77–3.74), the H-2 appearing as two dou-
−
1
−
33.7 KJ mol
and the value of entropy ob-
−
1
−1
tained from the intercept is 74.3 J mol
K
.
With the forming inclusion complex of Berb with
b-CD, the enthalpy of the system is largely de-
creased, which implies that the main force of the
inclusion reaction is the extrusion of ‘high energy
water’ from the cavity of b-CD upon the inclusion
complex formation. This is in agreement with the
conclusion reported by Nishijo et al. [21].

J.-S. Yu et al. / Spectrochimica Acta Part A 58 (2002) 249–256
255
blets (at l 3.56), and the H-4 triplet (at l 3.52).
The addition of Berb, in fact, influences the
chemical shifts of b-CD resonance (Fig. 7c1).
The H-3 and H-5 resonances are shifted upfield
by 0.02 and 0.01 ppm, respectively. The H-3
and H-5 protons are located in the interior of
the b-CD cavity, so that Berb comes into con-
siderable contact with the interior proton of b-
CD. This is in agreement with the observations
of Demarco and Thakkar [24], who reported a
strong shielding of protons inside the b-CD cav-
ity for inclusion complexes formed with aro-
matic substrates. Furthermore, The H-5 and H-6
resonances appear somewhat resolved with re-
spect to that in pure b-CD, suggesting that Berb
comes into close proximity with both the H-5
and H-6 protons.
H-12, H-14 upfield. The shifts are accompanied
by a loss in resolution of H-11 and H-12. The
results suggest that the interaction of Berb and
b-CD are presumably of a hydrophobic nature,
taking between the b-CD cavity and the Berb
molecule, with the hydrophilic part of Berb ex-
tending outside of the b-CD cavity. This is con-
sistent with the hydrophobic property of b-CD
cavity.
4
. Conclusion
The present study reveals that proper size
matching between the CD/Berb complex
strongly affects the stability of the inclusion.
Berb/a-CD complex gives equilibrium constant
smaller than that of Berb/b-CD complex. The
pH study gives a firm reason to include Berb
with b-CD, since pH 7.20 is a very close ap-
proximation to that of our body. The enthalpy
and entropy values show that release of ‘high
energy water’ molecules from the cavity in the
complex formation process is the main force,
while hydrogen binding, Van der Waals force
and hydrophobic interaction may also contribute
to the complex formation. For Berb molecule,
the dielectric constant of b-CD cavity is
analogous to absolute alcohol, that is m:24.
From the results of fluorescence, absorption and
NMR study, we illustrate the most probable
configuration for the Berb/b-CD complex as
Fig. 8 (not drown to scale), with the hydropho-
bic part of the Berb extending inside the cavity,
while the hydrophilic part leaving outside the
cavity. This approach of inclusion between b-
CD and Berb conforms with the result of
Schuette et al. [26], who reported that the pro-
tons of acridine could no longer rotate freely
and are specifically involved in the interaction
with b-CD.
1
Fig. 7b shows the H NMR spectra of Berb.
1
The assignments of Berb H NMR peaks are as
follows: the H-8, H-13, H-14, H-4, H-2, H-9%
and H-10% singlet located at l 9.52, 8.36, 7.37,
6
.83, 5.98, 3.99 and 3.98, respectively; the H-11
and H-12, appearing as doublets, located at l
.93 and 7.84, respectively; the H-6 unresolved
7
band located at 3.42, due to the effect of nitro-
gen positive ion; and the H-5 triplets located at
1
l 3.11. The protons of H nuclear magnetic res-
onance spectra of Berb molecule are in agree-
ment with the results reported by Jewers et al.
[
25]. Fig. 7c shows the chemical shifts of Berb
resonance in the presence of b-CD. It is interest-
ing to note that the chemical shifts of Berb res-
onance are influenced by the addition of b-CD.
Except for H-2, H-9% and H-10%, Berb resonance
spectra are shifted from 0.03 to 0.19 ppm, with
H-8, H-4, H-5, H-6 downfield and H-13, H-11,
Acknowledgements
This study was supported by the National
Science Foundation of China and the Subject
Development Project of Nanjing University.
Fig. 8. Schematic depiction of b-CD complex with berberine.

2
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