Voltammetric and spectrophotometric study on the complexation of glibenclamide with β-cyclodextrin

Article abstract of DOI:10.1007/s10847-010-9801-9

The formation of an inclusion complex of glibenclamide (GL) with β-cyclodextrin (β-CD) in an aqueous ethanolic buffer solution of pH 7.0 has been investigated by UV spectrophotometry and differential pulse voltammetry and its stability constant is determined to be 855 and 354.15 M^-1, respectively. The phase solubility profile, based on the spectrophotometric absorbance's variations, was classified as A_L-type, indicating the formation of 1:1 stoichiometric inclusion complex of glibenclamide with β-CD with a stability constant value, K_S, of 846 M^-1.

Full text of DOI:10.1007/s10847-010-9801-9

J Incl Phenom Macrocycl Chem (2010) 68:417–421
DOI 10.1007/s10847-010-9801-9
ORIGINAL ARTICLE
Voltammetric and spectrophotometric study on the complexation
of glibenclamide with b-cyclodextrin
•
Abd-Elgawad Radi Shimaa Eissa
Received: 14 January 2010 / Accepted: 4 May 2010 / Published online: 22 May 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The formation of an inclusion complex of gli-
benclamide (GL) with b-cyclodextrin (b-CD) in an aque-
ous ethanolic buffer solution of pH 7.0 has been
investigated by UV spectrophotometry and differential
pulse voltammetry and its stability constant is determined
to be 855 and 354.15 M-1, respectively. The phase solu-
bility profile, based on the spectrophotometric absor-
bance’s variations, was classified as AL-type, indicating the
formation of 1:1 stoichiometric inclusion complex of gli-
benclamide with b-CD with a stability constant value, KS,
agent widely-used to lower blood glucose levels in patients
with type II non-insulin-dependent diabetes mellitus. It acts
mainly by stimulating endogenous insulin release from
beta cells of the pancreas [3]. Glibenclamide has extremely
poor aqueous solubility and resulting low bioavailability.
Consequently, an improved biological performance of this
drug through enhancing its solubility and dissolution rate
by complexation with b-cyclodextrin and 2-hydroxypro-
pyl-b-cyclodextrin has been reported [4–6], and the
more recently; a study of the ability of hydroxybutenyl
b-cyclodextrin (HBenbCD) to form complexes with GL
with enhanced solubility has been reported [7].
of 846 M-1
.
Keywords Cyclodextrin ꢀ Glibenclamide ꢀ
Spectrophotometry ꢀ Voltammetry
In the present paper, the interaction of GL with b-CD
has been studied using voltammetric and spectrophoto-
metric methods. Furthermore, the stability constant of the
GL-b-CD complex was obtained from the decrease in the
peak current, or from the variation in the absorption spectra
and from the phase solubility diagram.
Cyclodextrins are cyclic organic compounds obtained by
enzymatic transformation of starch. Among the class of
‘‘host’’ molecules, the b-cyclodextrin (b-CD) is one of the
most abundant natural oligomers and corresponds to the
association of seven glucose units with cavity which
exhibits a hydrophobic character whereas the exterior is
strongly hydrophilic. In pharmaceutical industries, the
inclusion process of pharmaceutical molecules with b-CD
led to important modifications of pharmaceutical properties
of guest molecules, to enhance solubility, chemical stability,
and bioavailability of the substance [1, 2].
Experimental
Materials and reagents
GL powder of pharmaceutical purity grade was a generous
gift provided by Sanofi Aventis, Egypt. b-CD was pur-
chased from Sigma Chemical Company (St. Louis, USA).
Phosphate buffer (0.2 M, pH 7.0, from potassium dihy-
drogen phosphate KH2PO4 and disodium hydrogen phos-
phate Na2HPO4) was used. For the preparation of standard
GL stock solution (1.0 9 10-3 M), 5 mg of GL was
accurately weighted, dissolved in ethanol and then adjusted
to 10 mL with ethanol. All materials used without any
further purification and doubly distilled water were used
throughout the study.
Glibenclamide (GL) (1-[[p-[2-(5-chloro-o-anisami-
do)ethyl] phenyl]-sulfonyl]-3-cyclohexylurea; glyburide) is
a potent, second generation oral sulfonylurea antidiabetic
A.-E. Radi (&) ꢀ S. Eissa
Department of Chemistry, Faculty of Science (Dumyat),
Mansoura University, 34517 Dumyat, Egypt
e-mail: abdradi@yahoo.com
123

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J Incl Phenom Macrocycl Chem (2010) 68:417–421
the experimental data, this may suggest that the complex of
GL with b-CD is a 1:1 association complex.
Apparatus
The solubility diagram was obtained according to Higu-
chi and Connors [11]. Briefly, excess amounts of solid GL
(40 mg) were added to 10 mL flasks of aqueous solutions
(phosphate buffer pH 7.0: ethanol, 90:10 (v/v) containing
various concentrations of b-CD, 0.0 - 6.0 9 10-3 M). The
flasks were sealed and the suspensions were shaken for 24 h
at 25 °C. After equilibrium attainment for one weak, the
samples were filtered through a 0.45 lm membrane filter
and suitably diluted. The GL concentration in the filtrate was
spectrophotometrically analyzed at 302 nm. The stability
constant, KS, was calculated from the phase solubility dia-
grams according to the equation:
The UV spectra were performed by a Perkin Elmer UV–VIS
double beam spectrophotometer equipped with a PC for data
processing UV WinLab-ver 2.80.03, Perkin Elmer, USA).
The spectra were recorded over the wavelength range from
200to350 nmatascan speed of240 nm min-1. A quartz cell
with a 1.0 cm path length was used. All pH measurements
were performed on a CG 808 (Schott Gerate, Germany)
digital pH-meter with glass combination electrode.
The voltammetry experiments were performed using
CHI610C Electrochemical Analyzer controlled by CHI
Version 9.09 (USA). A three-electrode system was com-
posed of a glassy carbon (BAS model MF-2012, U = 3 mm)
working electrode, an Ag/AgCl/3 M KCl (BAS model
MF-2063) reference electrode and a platinum wire (BAS
model MW-1032) counter electrode. The working electrode
surface was polished with 0.3 and 0.05 lm alumina slurries
before each measurement.
Slope
KS ¼
ð3Þ
So ꢂ ð1 ꢁ SlopeÞ
where So is the solubility of glibenclamide in the absence
of b-CD and the slope means the corresponding slope of
the phase solubility diagrams, i.e., the slope of the GL
concentration versus b-CD concentration graph. The gli-
benclamide concentration was obtained using calibration
curve constructed in the same experimental conditions.
The calibration curve of GL was constructed using a
Methods
Cyclic voltammetry and differential pulse voltammetry
experiments were performed for 7.0 9 10-5 M of GL in
phosphate buffer (0.2 M, pH 7.0): ethanol, 90:10 (v/v)
containing various concentrations of b-CD (0.0–5.0 9
10-3 M). The current titration-equation was described as
follows [8]:
series of standard solutions in the range of (1.0 9 10-5
-
5.0 9 10-5 M) prepared by appropriate dilutions of the
stock solution of GL with phosphate buffer pH 7.0 in away
that the final solutions were composed of phosphate buffer:
ethanol, 90:10 (v/v).
ð1 ꢁ AÞ
1=CCD ¼ KS
ꢁ KS
ð1Þ
1 ꢁ i=io
Results and discussion
where CCD is the concentration of b-CD, KS is the apparent
stability constant, io and i are the peak current without and
with b-CD. A is the proportional constant. The condition of
using this equation is that a 1:1 association complex is
formed and CCD is much larger than the total concentration
of GL in solution. In other words, if Eq. 1 corresponds well
to the experimental data, this may suggest that the complex
of GL with b-CD is a 1:1 association complex.
The absorption spectra were recorded in the range of
200–350 nm, and for the calculation of stability constant,
the change of absorption of GL was measured at 302 nm as
a function of b-CD concentration. The concentration of
glibenclamide was fixed at 1.0 9 10-5 M and the b-CD
concentration was changed from 1.0 9 10-4 to 5.0 9 10-4
M. The stability constant can be evaluated spectrophoto-
metrically according to the following equation [9, 10]:
Electrochemical results
The electrochemical oxidation behaviour of glibenclamide
on a bare carbon paste electrode (CPE) and a Sephadex-
modified carbon paste electrode (SMCPE) has been pre-
viously reported [12], the cyclic voltammetric behavior of
the compound yielded one oxidation process with one
electron-one proton transfer, probably by the formation of a
cation radical at the nitrogen of the amide group. However,
no previous electrochemical data were available concern-
ing the electrochemical oxidation behaviour of glibencla-
mide on a glassy carbon electrode.
As can be seen in Fig. 1, the cyclic voltammetric
behaviour of 7.0 9 10-5 M glibenclamide in the absence
and presence of b-CD yielded one oxidation process in
phosphate buffer pH 7.0. On the reverse sweep, no distinct
reduction wave was observed, indicating that the drug is
irreversibly oxidized at the glassy carbon electrode. The free
glibenclamide gave anodic peak potential at 1.26 V, which
reflects the oxidation of the amide group, the most probable
A0
eG
eG
1
¼
þ
ð2Þ
A ꢁ A0 eHꢁG ꢁ eG eHꢁG ꢁ eG KSCCD
where A0 and A are the absorbances of the free guest and the
apparent one, eG and eCD–G are the absorption coefficients of
the guest and complex, respectively. Thus, if Eqs. 1 and 2 fit
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J Incl Phenom Macrocycl Chem (2010) 68:417–421
419
methoxyphenyl residue of glibenclamide molecule in the
2-hydroxypropyl- b-cyclodextrin cavity, while the sulpho-
nylurea group remains out side [6]. On the other hand, the
decrease of the peak current can be ascribed as a diminution
of the apparent diffusion coefficient of the glibenclamide
included in the complex with b-CD, when compared with the
apparent diffusion coefficient of glibenclamide alone.
The CV peak currents were proportional to the square
root of scan rates in the range of 10–100 mVs-1 both
without and with b-CD. The slope of the linear plot of ip
versus m1/2 without b-CD (0.3651 lA mV-1/2 s1/2) was
more than that with b-CD (0.20293 lA mV-1/2 s1/2) as
shown in Fig. 1 as inset, suggesting that the diffusion
coefficient of the free form of glibenclamide was larger than
that of the complexed form of glibenclamide with b-CD.
The inclusion phenomenon of glibenclamide with b-CD
was also studied by differential pulse voltammetry, which
is a more sensitive electrochemical method (Fig. 2). The
differences in the peak currents using DPV are more pro-
nounced, obtaining more accurate results for calculation
of the stability constant. According to the decrease of
peak currents with increasing concentration of b-CD, the
following equation was obtained: 1/CCD = 134.3/(1 - i/
io) - 354.15 with a linear correlation coefficient (r) of
0.9996. This revealed that the inclusion complex of gli-
benclamide with b-CD was a 1:1 association complex and
the stability constant (KS), which is usually used to char-
acterize the inclusion phenomena of cyclodextrin systems
[14], was 354.15 M-1 as calculated from the y-intercept.
3.5
2.8
2.1
1.4
0.7
0.0
14
12
10
8
(a)
(b)
6
2
4
6
8
10
1
-1 1/2
v1/2(mVs )
4
2
2
0
-2
0.6
0.8
1.0
1.2
1.4
1.6
E/V vs Ag-AgCl
Fig. 1 Cyclic voltammograms for 7.0 9 10-5 M glibenclamide solu-
tion obtained in phosphate buffer (pH 7.0) using a scan rate of
50 mVs-1. (1) Without b-CD (2) with 5.0 9 10-3 M b-CD. Inset is the
plot of ip versus m1/2 (a) without b-CD, (b) with 5.0 9 10-3 M b-CD
oxidation centre in the glibenclamide molecule. This oxi-
dation potential is in agreement with the reported oxidation
potentials of propanil and related N-substituted amides [13],
which confirms that oxidation occurs at the nitrogen of the
amide group as previously reported at the carbon paste
electrode [12]. The addition of b-CD to the solution of
glibenclamide causes two main changes in the voltammo-
grams. Firstly, the anodic peak potential (Ep) shifted to a
more positive direction and secondly, the peak currents (ip)
decreased. These results can be ascribed to the formation of
the inclusion complexes according to the equilibrium:
Spectrophotometric studies
GL þ b-CD ꢀ GL=b-CD
wherein GL/b-CD means the inclusion complex between GL
and b-CD. The positive shift in the EP reveals that the amide
groups in the glibenclamide molecules were oxidized with
more difficulty, suggesting that they were included in the
b-CD cavity, while the sulphonylurea groups remain out side
(Scheme 1). Otherwise, if the other side of the glibenclamide
was included in the b-CD cavity there would not be any
change in the peak potential. However, the proposed inclu-
sion mechanism is in agreement with that reported for the
inclusion complex of glibenclamide with 2-hydroxypropyl-
b-cyclodextrin, in which the observations suggest that
the complex is formed by inclusion of the 3-chloro-6-
The formation of inclusion complex between GL and b-CD
could be further confirmed by a spectroscopic experiment.
The absorption spectra of GL in the absence, and presence, of
b-CD are shown in Fig. 3. It is noticed that upon addition of
b-CD, the absorption maximum of glibenclamide at 302 nm
has a hypsochromic shift (i.e., shifted towards lower wave-
length). However, the UV–vis absorbance decreased with
increasing concentration of b-CD. The spectral data further
proved the formation of the inclusion complex of GL with
b-CD, and the stability constant, KS, of this complex can be
determined according to Benesi–Hildebrand equation [15],
under the condition of the 1:1 inclusion complex formation
Scheme 1 The proposed reaction sketch of the inclusion complex of glibenclamide with b-CD
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J Incl Phenom Macrocycl Chem (2010) 68:417–421
(M) and correlation coefficient of 0.998 and Fig. 5 shows
2.0
1.6
1.2
0.8
0.4
0.0
the phase solubility diagram of GL with b-CD with a
1000
750
500
250
0
1
linear regression equation of C(M) = 4.239 9 10-5
0.03478CCD, and correlation coefficient of 0.991.
?
5
The phase solubility diagrams of GL in aqueous solutions
of b-CD obtained at 25 °C shows that the solubility of GL
increased linearly as a function of b-CD concentration and
over the range of concentrations studied showed the features
of an AL-type following Higuchi and Connors’ classification
[11]. The increase in the solubility can be attributed to the
formation of inclusion complexes between GL and the b-CD
characterized by greater solubilities than that of GL alone.
Fig. 5 shows that in the absence of b-CD, the solubility of GL
3
4
5
6
7
8
9 10
1/ (1-i/i0)
0.6
0.8
1.0
1.2
1.4
1.6
E/V vs Ag-AgCl
0.4
Fig. 2 DPV curves for 7.0 9 10-5
obtained in phosphate buffer (pH 7.0) in absence (1) and presence
M
glibenclamide solution
0.18
0.16
0.14
of (2) 1.0 9 10-3, (3) 3.0 9 10-3, (4) 4.0 9 10-3, (5) 5.0 9 10-3
b-CD. Inset is the plot of 1/CCD versus 1/(1 - i/io)
M
0.3
0.12
0.10
0.08
0.05
-5
0.2
0.1
0.0
0.06
0.00002 0.00003 0.00004 0.00005
-10
0.04
C(mol/l)
1
4
-15
-20
-25
0.03
0.02
0.01
0.00
2000 4000 6000 8000 10000
1/CCD
260
280
300
320
340
360
Wavelength (nm)
Fig. 4 Absorption spectra of various concentrations of glibenclamide
under the conditions of phosphate buffer pH 7.0: ethanol, 90:10 (v/v).
Inset is the calibration curve
260
280
300
320
340
360
Wavelength(nm)
Fig. 3 Absorption spectra of glibenclamide (1.0 9 10-5 M) in the
absence and presence of various concentrations of b-CD: (1) 0, (2)
1.0 9 10-4, (3) 3.0 9 10-4, (4) 5.0 9 10-4 M. Inset is the plot of Ao/
(A - Ao) versus 1/CCD
equilibrium with an excess concentration of b-CD compared
to the total concentration of GL. According to Eq. 2, from an
Ao/(A - Ao) versus 1/CCD plot (inset of Fig. 3), the follow-
ing equation was obtained: Ao/(A - Ao) = - 1.89873 -
0.00222/CCD with a linear correlation coefficient (r) of
0.996. The ratio of the intercept to the slope gives the value of
stability constant of 855 M-1
.
Phase solubility studies
Figure 4 shows the absorption spectra of glibenclamide
without b-CD and shows as inset the calibration curve with
a linear regression equation of A = -0.00901 ? 3553.3C
Fig. 5 Phase solubility diagrams for glibenclamide with increasing
concentration of b-CD established by UV–Vis method under the
condition of phosphate buffer pH 7.0: ethanol, 90:10 (v/v)
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J Incl Phenom Macrocycl Chem (2010) 68:417–421
421
is 4.24 9 10-5 M while in presence of b-CD, the solubility
increases to 2.6 9 10-4 M at the maximum concentration of
the b-CD studied. As the slope of the solubility curves is less
than unity, it can be assumed that the stoichiometry of
inclusion complexes is 1:1. The stability constants of the
inclusion complexes were calculated from the straight-line
diagram according to the Eq. 3. We have obtained a KS value
of 846 M-1 for the inclusion complex between GL and
b-CD, suggesting a relatively stable complex. The obtained
KS value was lower than the published value [16], perhaps
due to addition of ethanol, which led to decrease in the
polarity of the medium, and this is unfavorable for the
hydrophobic interaction between GL and b-CD.
In our experiments, we can notice that the electrochemical
and spectrophotometric methods do not give close results for
the stability constants. The probable reason could be the use
of different techniques for the calculation of the stability
constant as it is a fact that the stability constants of the host–
guest molecules significantly depends upon the technique
used for their evaluation [17–19]. The spectrophotometric
and the phase solubility methods depending on the same
technique give the close results for the stability constant (855
and 846 M-1), while the voltammetric method depending on
measuring another experimental property (changes of the
peak current) doesn’t give the same result (354 M-1).
2. Loftsson, T., Brewster, M.: Pharmaceutical applications of
cyclodextrins. J. Pharm. Sci. 85, 1017–1025 (1996)
3. Ashcroft, F.M.: Mechanisms of the glycaemic effects of sulfo-
nylureas. Horm. Metab. Res. 28, 456–463 (1996)
4. Babu, R.J., Pandit, J.K.: Enhancement of dissolution rate and
hypoglycemic activity of glibenclamide with b-cyclodextrin. STP
Pharma Sci. 5, 196–201 (1995)
5. Sanghavi, N.M., Tandel, V., Venkatesh, H.: Solubilization of
glibenclamide with b-cyclodextrins & its derivatives. Drug. Dev.
Ind. Pharm. 20, 1275–1283 (1994)
´
6. Esclusa-Dıaz, M.T., Torres-Labandeira, J.J., Kata, M., Vila-Jato,
J.L.: Inclusion complexation of glibenclamide with 2-hydroxy-
propyl-b-cyclodextrin in solution and in solid state. Eur. J.
Pharm. Sci. 1, 291–296 (1994)
7. Klein, S., Wempe, M.F., Zoeller, T., Buchanan, N.L., Lambert,
J.L., Ramsey, M.G., Edgar, K.J., Buchanan, C.M.: Improving
glyburide solubility and dissolution by complexation with hy-
droxybutenyl-b-cyclodextrin. J. Pharm. Pharmacol. 61, 23–30
(2009)
8. Zhao, G.C., Zhu, J.J., Zhang, J.J., Chen, H.Y.: Voltammetric
studies of the interaction of methylene blue with DNA by means
of b-cyclodextrin. Anal. Chim. Acta 394, 337–344 (1999)
9. Dang, X.J., Tong, R., Li, H.L.: The electrochemistry of the
inclusion complex of anthraquinone with b-cyclodextrin studied
by means of OSWV. J. Inclusion Phenom. 24, 275–286 (1996)
10. Ni, M.Y., Wang, Y., Li, H.L.: Electrochemical and spectral
properties of phenylhydrazine in the presence of b-cyclodextrin.
Pol. J. Chem. 71, 816–822 (1997)
11. Higuchi, T., Connors, K.A.: Phase-solubility techniques. Adv.
Anal. Chem. Instrum. 4, 117–212 (1965)
12. Radi, A.E.: Voltammetric study of glibenclamide at carbon paste
and Sephadex-modified carbon paste electrodes. Anal. Bioanal.
Chem. 378, 822–826 (2004)
13. Garrido, E.M., Lima, J.L.F., Delerue-Matos, C., Borges, F., Silva,
A.M.S., Oliveira Brett, A.M.: Electrochemical oxidation of
propanil and related N-substituted amides. Anal. Chim. Acta 434,
35–41 (2001)
14. Taraszewska, J., Piasecki, A.K.: Inclusion complexes of isomeric
chloronitrobenzenes with a- and b-cyclodextrins studied by
polarography: analysis of the possibilities of the method.
J. Electroanal. Chem. 226, 137–146 (1987)
15. Benesi, H.A., Hildebrand, J.H.: A spectrophotometric investiga-
tion of the interaction of iodine with aromatic hydrocarbons.
J. Am. Chem. Soc. 71, 2703–2707 (1949)
16. Zerrouk, N., Corti, G., Ancillotti, S., Maestrelli, F., Cirri, M.,
Mura, P.: Influence of cyclodextrins and chitosan, separately or in
combination, on glyburide solubility and permeability. Eur. J.
Pharm. Biopharm. 62, 241–246 (2006)
Conclusion
The effect of b-CD on the voltammetric behavior of gli-
benclamide showed a positive shift in the anodic peak
potential and a decrease in the peak current. From these
changes, we can assume that the amide group on the gli-
benclamide molecule was located inside the cavity of b-CD
and a diminution of the peak current due to a diminution in
the diffusion coefficient of glibenclamide as a consequence
of the formation of an inclusion complex with b-CD. The
phase solubility diagrams of GL in aqueous solutions of
b-CD showed that the solubility of GL increased linearly as
a function of b-CD concentration. The increase in solu-
bility can be attributed to the formation of inclusion
complexes between GL and the b-CD characterized by
greater solubilities than that of GL alone. From the vol-
tammetric, spectrophotometric and phase solubility results,
it may be concluded that b-CD forms 1:1 type inclusion
complexes with glibenclamide and the obtained stability
constants were 354, 855 and 846 M-1, respectively.
17. Junquera, E., Pena, L., Aicart, E.: A conductimetric study of the
interaction of b-cyclodextrin or hydroxypropyl-b-cyclodextrin
with dodecyltrimethylammonium bromide in water solution.
Langmuir 11, 4685–4690 (1995)
18. Junquera, E., Pena, L., Aicart, E.: Micellar behavior of the
aqueous solutions of dodecylethyldimethylammonium bromide.
A
characterization study in the presence and absence of
ˆ
hydroxypropyl-a-cyclodextrin. Langmuir 13, 219–224 (1997)
19. Satake, I., Ikenoue, T., Takeshita, T., Hayakawa, K., Maeda, T.:
Conductometric and potentiometric studies of the association of
a-cyclodextrin with ionic surfactants and their homologs. Bull.
Chem. Soc. Jpn. 58, 2746–2750 (1985)
References
1. Challa, R., Ahuja, A., Ali, J., Khar, R.K.: Cyclodextrins in drug
delivery: an updated review. AAPS Pharm. Sci. Tech. 6, E329–
E355 (2005)
123