Host-guest system of Vitamin B10 in β-cyclodextrin: Characterization of the interaction in solution and in solid state

Article abstract of DOI:10.1007/s10847-010-9763-y

Conventional drugs are usually formulated for the immediate release of the medicinal substances and for obtaining the desired therapeutic effect. The aim of this paper was to investigate the possible interactions between Vitamin B10 and β-cyclodextrin (β-

Full text of DOI:10.1007/s10847-010-9763-y

J Incl Phenom Macrocycl Chem (2010) 68:175–182
DOI 10.1007/s10847-010-9763-y
ORIGINAL ARTICLE
Host–guest system of Vitamin B10 in b-cyclodextrin:
characterization of the interaction in solution and in solid state
•
Irina Kacso Gh. Borodi S. I. Farcas
•
•
´
A. Hernanz I. Bratu
•
Received: 23 October 2009 / Accepted: 24 February 2010 / Published online: 12 March 2010
Ó Springer Science+Business Media B.V. 2010
Abstract Conventional drugs are usually formulated for
the immediate release of the medicinal substances and for
obtaining the desired therapeutic effect. The aim of this
paper was to investigate the possible interactions between
Vitamin B10 and b-cyclodextrin (b-CD), to determine the
physical-chemical characteristics and the interactions
present in the corresponding inclusion compound. The so-
obtained compounds were characterized by X-ray diffrac-
Keywords Inclusion compound ꢀ p-Amino benzoic acid ꢀ
Cyclodextrin ꢀ Molecular spectroscopy (FTIR, UV–vis,
1
fluorescence, H NMR) ꢀ DSC ꢀ X-ray powder diffraction
Introduction
A series of researches will conduct in the field of controlled
drug release (release of Vitamin included in cyclodextrin).
Para-amino benzoic acid (PABA) or Vitamin B10 (see
Fig. 1a), is an intermediate in bacterial synthesis of folate
[1], the microorganisms in the intestines are capable of
synthesizing folic acid (an important factor in the protein
use) from this compound, and humans lack this ability.
Vitamin B10 is sometimes marketed as an essential nutri-
ent for use whenever normal Vitamin B10 synthesis by
intestinal bacteria is insufficient. The compound is used as
a UV filter in sunscreen formulations [2], as a drug against
fibrotic skin disorders, in treating irritable bowel syndrome
[3, 4].
1
tion, DSC and FTIR spectroscopy. H NMR and UV–vis
spectroscopic methods were employed to study the inclu-
sion process in aqueous solution. The X-ray powder dif-
fraction patterns demonstrate the inclusion compound
formation, especially for the lyophilized product where the
amorphous phase dominates. The existence of the inclusion
compounds obtained by different methods was confirmed
by comparing with DSC and FTIR data of the pure com-
pounds and the (1:1) Vitamin B10:b-CD physical mixture
(pm). 1H NMR measurements on aqueous solutions of
Vitamin B10 and b-CD in D2O allowed us to establish the
corresponding Vitamin B10’s and cyclodextrin’s protons
implied in the complexation process. 2D NMR spectros-
copy established the geometry of the inclusion complex. 1H
NMR, UV–Vis and fluorescence data were used to obtain
the stoichiometry and the stability constant of the complex.
CDs are cyclic oligosaccharides consisting of six, seven
or eight units of a-D-(?)-glucopyranose, referred to as a-,
b- and c-CD respectively [5] (see Fig. 1b) obtained from
starch by enzymatic reaction, that may encapsulate a wide
variety of guest molecules (completely, or at least partially)
in their hydrophobic cavity.
Through controlled release, these systems ensure control
of the release and of the absorption of the medicinal sub-
stances from the respective system.
´
I. Kacso ꢀ Gh. Borodi ꢀ S. I. Farcas ꢀ I. Bratu (&)
The aim of this paper was to obtain an inclusion com-
pound (IC) of Vitamin B10 with b-cyclodextrin (b-CD)
and to characterize it by FT IR, X-ray powder diffraction,
DSC, 1H NMR, UV–Vis and spectrofluorimetry. Molecular
modeling technique was employed to obtain the spatial
architecture of this supramolecular assembly.
National Institute for R&D of Isotopic and Molecular
´
Technologies Donath, 65-103 Cluj-Napoca, Romania
e-mail: ibratu@gmail.com
A. Hernanz
Depto de CC y TT Fısico Quimicas, UNED Madrid,
´
Senda del Rey 9, 28040 Madrid, Spain
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J Incl Phenom Macrocycl Chem (2010) 68:175–182
substance was constant (10-4 mol/L) and the b-cyclodex-
trin concentration was increased progressively (10-3
–
10-2 mol/L) (pH % 5). The solutions were keept for 12 h
at room temperature in order to equilibrate them.
Fluorescence spectroscopy measurements were per-
formed using a 1.0 cm quartz cell with a JASCO FP6500
spectrofluorimeter. The excitation wavelength of 266 nm
was employed, and the emission spectra were recorded
from 300 to 450 nm and 600 to 730 nm, respectively. The
Vitamin B10 concentration was fixed at 0.05 mM while the
CDs concentration was varied from 0 to 8 mM. All aque-
ous solutions were shaken for 12 h.
Fig. 1 a p-Aminobenzoic acid (PABA, Vitamin B10); b cyclodex-
trins: for b-CD, N = 7
1H NMR spectra were obtained with Bruker Avance 500
spectrometer after at least 15 min of thermal equilibration
at 25 °C. The spectrometer was operated at
500.1325 MHz, with the following parameters: 32,000 data
points, pulses of 90°, 2 s delay between scans (16 scans)
and a digital resolution of 0.588 Hz/point. The chemical
shifts were expressed in parts per milion (ppm) relativ to
the chemical shift of HOD signal located at 4.6897 ppm.
The inclusion complexes of b-CD (host, H) with the active
substance (G) were prepared in aqueous solutions starting
from a ‘‘mother’’ millimolar concentration of H and G in
D2O. b-CD (having B 15% water in weight) was obtained
from CYCLOLAB Hungary being used without further
purification. The aqueous solutions were prepared taking
into account the molecular water present in b-CD. Two
starting solutions, of b-CD and of guest molecule, of
10 9 10-3 mol/dm3 concentration in D2O (ROMAG
Turnu Severin, Romania), were prepared. Starting from
these ‘‘mother’’ solutions, several mixtures having different
molar ratios at constant volume of G and b-CD were pre-
pared. The sum of the total concentration M = ([b-
CD]t ? [G]t) (where the total index refers to the total
concentration, being equal with 10 9 10-3 mol/dm3. The
molar ratio of the guest G, rG = [G]t/([b-CD]t ? [G]t)
varies between 0 and 1 with an interval of 0.1.
Experimental
The solid state inclusion compounds were prepared in
1:1 molar ratio of the b-cyclodextrin (host) and Vitamin
B10 (guest). The kneaded compound (kn) was prepared by
mixing in an agate mortar the mixture of both components,
using distilled water, for 1 h. The co precipitated product
(co) was prepared by mixing the equimolar aqueous solu-
tion of the Vitamin B10 and b-cyclodextrin, and stirring
this mixture for 24 h at room temperature. An identical
solution was frozen, and then freeze-dried in an Alpha 1-2
LD type freeze dryer to obtain the lyophilized product (fd).
FTIR measurements were performed with a FTIR
JASCO 6100 spectrometer in the in 4,000–400 cm-1
spectral range with a resolution of 4 cm-1 using KBr pellet
technique.
X-ray powder diffraction patterns were collected with
Bruker D8 Advance diffractometer in the 2h = 2–50°
angular domain using Cu Ka1 radiation. In order to
increase the resolution a monochromator was used to
eliminate Ka2 radiation.
DSC thermograms were registered with a DSC60 Shi-
madzu differential scannning calorimeter by heating the
samples from room temperature up to 350 °C, in a crimped
aluminium pan, under flowing nitrogen flux, the heating
rate being 10 °C/min. For data collection the Shimadzu
TA-WS60 and TA60 2.1 software were employed.
UV–vis spectra were registered with a double beam
JASCO V-550 spectrophotometer in the 190–400 nm
spectral range with a resolution of 0.2 nm. The absorption
spectra were obtained using 1 9 1 9 4 cm3 quartz cells.
The spectrophotometer was equipped with a thermostat
unit, the temperature in the cells was maintained at 20°C,
during the experiments. In the reference cell the employed
solvent was introduced, in the same volume ratio as the
used for solving the samples. The solutions were prepared
in bidistiled water, the concentration of the bioactive
The two-dimensional NMR spectrum at 500 MHz was
obtained on a solution with rG = 0.7 through standard
Bruker software. The conditions for ROESY phase-sensi-
tive spectra via time proportional phase incrementation
(TPPI) were: presaturation of residual HDO signal, spectral
widths of 6.8 ppm in both dimensions with a resolution of
0.83 and 1.66 Hz/point in f2 and f1 respectively and a
mixing time of 200 ms. The experiment was performed
using 4,096 data points in f2 and 2,048t1 increments with
16 scans per t1 value and a relaxation period of 2 s. A sine
function (SSB = 2) was applied in f1 and f2 before Fourier
transformation.
Molecular modeling computations were performed
using MM? molecular mechanics (Hyperchem software).
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J Incl Phenom Macrocycl Chem (2010) 68:175–182
177
Results and discussion
inclusion compound was obtained for the co product, see
Fig. 3. For co (co precipitated one) inclusion compound a
new crystalline compound was obtained having a crystal-
line structure distinct of that for the initial pure compounds.
The positions of the diffraction lines for the co inclusion
compound are different as compared to the corresponding
ones for Vitamin B10 and b-CD, i.e. the parameters of the
elementary cell for the new compound are different as
compared with the initial compounds’ ones.
FTIR
The following vibrational modes of Vitamin B10 molecule
(assigned elsewhere [6]) were affected (see Fig. 2) by
complexation process:
–
mas(C=O) located at 1,680 and 1,662 cm-1 in pure drug
spectrum is shifted to 1,685 cm-1 in the spectra of
inclusion compounds obtained by different methods. A
similar effect was observed by the analysis of a co
product spectrum [7]. The splitting of the mas(C=O)
band can be assigned to the coexistence of different
association type species present in solid state. The
higher frequency shift can be ascribed to the destruc-
tion of strong hydrogen bonding structure in uncom-
plexed drug upon inclusion compound formation with
b-CD.
DSC
DSC reveals some information on solid-state interactions
between drug and cyclodextrin. The DSC thermograms of
pure components and of Vitamin B10—b-cyclodextrin
inclusion compounds are presented in Fig. 4. The curve for
the b-cyclodextrin revealed a broad endothermic signal
from 74 to 118 °C, with DH = 200 kJ/mol, that corre-
sponds to the loss by evaporation of the water molecules
existing as residual humidity (t \ 100 °C) as well as those
included in the cavity (t [ 100 °C) [9, 10]. From 290 °C
onwards there is a new endothermic succeeded of the
exothermic, corresponding to the melting, respectively the
degradation of the b-cyclodextrin.
–
ds(NH2) vibrational mode, centered on 1,635 cm-1 in
pure drug spectrum, is shifted to 1,630 cm-1 in the
spectra of inclusion compounds obtained by different
methods.
These two vibrational bands changes certify the inclu-
sion compound formation and offer an idea about the
mechanism of the inclusion compound formation.
The DSC curve of Vitamin B10 presents a sharp endo-
thermic peak at 191 °C, with DH = 15 kJ/mol, corre-
sponding to the melting of the drug, following the
degradation of the substance. In the case of the physical
mixture and the inclusion compounds of Vitamin B10 with
b-cyclodextrin obtained by kneading, co precipitation and
freeze-drying methods, the strong decreasing of dehydra-
tion endothermic peak of cyclodextrin was observed, as
well as a disappearance of the melting peak of the Vitamin
B10 in the inclusion compounds. Around 270 °C the
decomposition process begins [7]. The DSC thermogram
for the physical mixture shows a broad endothermic from
63 to 123 °C, with DH = 78 kJ/mol, from 265 °C begin-
ning the decomposition process. The thermogram of the
kneaded inclusion compound presents a small broad
endothermic from 56 to 120 °C, with DH = 66 kJ/mol, the
decomposition beginning from 276 °C. The inclusion
compound obtained by co precipitation shows a small
endothermic from 58 to 117 °C, DH = 93 kJ/mol and the
start of decomposition increased at 284 °C. The thermo-
gram of the freeze-dried inclusion compound shows a very
small broad endothermic from 60 to 105 °C with
DH = 50 kJ/mol, followed by a decomposition exothermic
started at 288 °C. The decreasing of dehydration endo-
thermic peak of cyclodextrin, the disappearance of Vitamin
B10 melting peak and the increasing of the decomposition
temperature are the indications for the inclusion process
between Vitamin B10 and b-cyclodextrin.
XRD
The crystal structure of the Vitamin B10 was already
reported [8]. For kn (kneaded product) and fd (freeze-dried
product) inclusion compounds products, mixtures of
amorphous and crystalline phases with very small crystal-
lites were obtained, whereas a new quite crystalline
Fig. 2 FTIR spectra of Vitamin B10 and of inclusion compound with
b-CD, 1,800–1,300 cm-1 spectral range
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J Incl Phenom Macrocycl Chem (2010) 68:175–182
Fig. 3 X-ray powder
diffraction patterns of b-CD,
Vitamin B10 and of the
inclusion compounds obtained
by kneading (kn),
coprecipitation (co) and freeze-
drying (fd)
Fig. 5 The chemical shifts in the 1H NMR spectra of cyclodextrin
(b-CD), Vitamin B10 and of the inclusion complex (IC) formed at
r = 0.5, 7.9–6.6 ppm domain
Fig. 4 DSC thermograms of Vitamin B10, b-cyclodextrin and of the
corresponding inclusion compounds
1H NMR
p-Amino benzoic acid has seven protons in its structure;
among them, four aromatic protons equivalent each two
(H2–H6; H3–H5), see Fig. 5, present two signals as two
1
doublets, at 7.63, respectively at 6.56 ppm in the H NMR
spectrum [11].
Fig. 6 The chemical shifts in the 1H NMR spectra of cyclodextrin (b-
CD), Vitamin B10 and of the inclusion complex (IC) formed at
r = 0.5, 4–3.4 ppm domain
The observed changes in the chemical shifts values of
the Vitamin B10 and of b-CD as compared to the chemical
shifts corresponding to the inclusion complex (see Figs. 5,
6 and Table 1) suggests a total inclusion of the guest
molecule in the cyclodextrin cavity.
B10—b-CD complex in D2O are presented in the
Figs. 7 and 8.
In order to establish the orientation of the Vitamin B10
inside CD cavity, 2D NMR experiments were performed.
Two portions of the 2D ROESY spectrum for the Vitamin
The cross-peaks between the aromatic hydrogens of
Vitamin B10 (located at 6.83 and 7.79 ppm) and the
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J Incl Phenom Macrocycl Chem (2010) 68:175–182
179
Table 1 The chemical shifts of
Proton
Vitamin B10 d (ppm)
b-CD d (ppm)
IC (1:1) d (ppm)
Dd (ppm)
the protons belonging to
Vitamin B10, b-cyclodextrin
and to the inclusion complex
(IC)
H2,6
7.8047
6.8565
7.7619
6.7935
3.8307
3.6850
3.5829
3.8054
0.0428
0.063
H3,5
H30
H50
H20
H60
3.9183
3.8096
3.6043
3.8331
0.0876
0.1246
0.0214
0.0277
By using the well-known Job plots for different protons
of Vitamin B10 and of b-CD, an 1:1 stoichiometry of the
inclusion complex was obtained, see Fig. 9.
UV–Vis
The UV–vis absorption spectra of Vitamin B10 are influ-
enced by the solvent nature and by the pH of the solution
[12]. By increasing the solution pH, the absorption spectra
present a blue shift. At pH = 1, when protonated form
dominates in the solution, kabs ꢀ 282 nm; by increasing pH
values till 4–5.5, then the deprotonation of the carboxyl
group process begins. Besides neutral species, monoanions
appeared which determined a decrease of the absorption
maximum till a 265 nm value for the wavelength [13]. The
absorption and emission spectra of Vitamin B10 in aqueous
solutions, at different pH values and various b-CD con-
centrations were also studied. At pH = 1 value Vitamin
B10 exists as monocation in aqueous solutions, presenting
an absorption maximum at 270 nm whereas at pH = 7
when the substance exists as monoanion in aqueous
Fig. 7 A section of 500 MHz 2D ROESY symmetrized spectrum of
Vitamin B10—b-CD complex showing the interaction between H2,6
of Vitamin B10 and H30, H50 of b-CD
Fig. 8 A portion of 500 MHz 2D ROESY symmetrized spectrum of
Vitamin B10—b-CD complex showing the interaction between H3,5
of Vitamin B10 and H30, H50 of b-CD
hydrogens H30 and H50 (3.77 and 3.63 ppm) indicate that
H2,6 (see Fig. 1) interact with H30 and H50 whereas the
hydrogens H3,5 interact only with H30. One can conclude
that Vitamin B10 molecule enters CD cavity with the
carboxylic group near to its narrower rim. This result is in
agreement with the data published before [12].
Fig. 9 Job plots for different protons of Vitamin B10 and b-CD
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J Incl Phenom Macrocycl Chem (2010) 68:175–182
solution, the absorption peak is located at 285 nm [12, 14].
For both situations an isosbestic point is observed. When
b-CD is present a weak blue shift (271–269 nm) is
observed at pH = 1 and a red shift (266–284 nm) at
pH = 7. The absorption intensity increases as the b-CD
concentration is increased.
Table 2 Batochromic shift of the absorption maximum
[b-CD] (mM)
k (nm)
A (u.a.)
8
286
284
282
278
271
267
266
0.888
0.876
0.846
0.782
0.768
0.758
0.776
7
6
5
By measuring the absorbance of some aqueous solutions
of Vitamin B10 (5 9 10-5 mol/L) and b-CD (10-4
–10-3 mol/L) at pH & 5 a batochromic shift is observed,
see Fig. 10 and Table 2, as compared to that for pure
Vitamin B10 (266 nm). The appearance of two isosbestic
points is also observed, at 243 and 269 nm.
4
1.5
0.5
Fluorescence
The fluorescence of the Vitamin B10 depends on pH value,
also: at acid pH value small changes of the emission
spectra (with maximum at 350 nm) are observed whereas
at pH = 7 a small red shift (340–347 nm) is observed [12].
A diminishing of the intensity as the b-CD quantity is
increased is also observed. The red shift of the emission
maximum suggests a localization of the COOH group
inside b-CD cavity and also the interaction of the NH2
group of the Vitamin B10 molecule with the OH groups of
the b-CD.
The fluorescence measurements were done with a set of
aqueous solutions similar to those used for UV–vis
absorption ones. The excitation was performed at 269 nm
isosbestic point. Two emission maxima were obtained, at
338 and 662 nm. The second maximum can be assigned to
the existence of intermolecular hydrogen bridges [15].
By increasing the b-CD excess, an intensity decreasing
of the emission maximum and a red shift are observed. At a
certain excess level both emission intensity and maximum
wavelength remain constant, see Fig. 11 and Table 3.
Fig. 11 Fluorescence spectra of Vitamin B10 and the inclusion
complexes with b-CD
Table 3 Batochromic shift of the emission maximum
Sample
k (nm)
F
[Vit.B10] (0.05 mM)
338
339
340
341
342
343
343
343
344
344
344
201
156
124
105
100
97
[Vit.B10] ? [b-CD] (0.5 mM)
[Vit.B10] ? [b-CD] (1 mM)
[Vit.B10] ? [b-CD] (1.5 mM)
[Vit.B10] ? [b-CD] (2 mM)
[Vit.B10] ? [b-CD] (2.5 mM)
[Vit.B10] ? [b-CD] (3 mM)
[Vit.B10] ? [b-CD] (4 mM)
[Vit.B10] ? [b-CD] (5 mM)
[Vit.B10] ? [b-CD] (6 mM)
[Vit.B10] ? [b-CD] (7 mM)
93
92
86
85
85
The decreasing of the fluorescence intensity and the blue
shifted fluorescence maximum suggest the formation of an
inclusion complex between Vitamin B10 and b-CD. The
linearity in the plot reveals the formation of 1:1 complex
between Vitamin B10 and b-CD (Fig. 12). The stability
Fig. 10 UV–Vis spectra of the inclusion complexes of Vitamin B10
with b-cyclodextrin ([B10] = 0.05 mM, [b-CD] = variable)
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J Incl Phenom Macrocycl Chem (2010) 68:175–182
181
constant K can be determined [11, 16] by using Benesi–
Hildebrand equation:
1
1
1
¼
þ
F
F ꢁ F0 KðF1 ꢁ F0Þ½b-CDꢂ0
1 ꢁ F0
where: K, the inclusion constant; F0, the fluorescence
intensity of Vitamin B10 without b-CD; F, the fluorescence
intensity with b-CD; F?, the fluorescence intensity of
Vitamin B10 with the highest concentration of b-CD.
The association constant, obtained from the first emis-
sion maximum, is 986 M-1 (log K & 2.99). This value is
comparable with the ones obtained by 1H NMR (2.8 0.6)
[11] or circular dichroism (2.7) [17].
Molecular modeling
Molecular mechanics computations have been carried out
with the HyperChem software [18, 19] to optimize the
geometry of p-amino benzoic acid and b-CD in vacuum.
The b-CD model was taken from CSD Entry with ref. code
BCDEXD03 [20]. p-Amino benzoic acid molecule was
obtained from geometrical optimization of the HyperChem
software. In the starting models the p-amino benzoic acid
molecules were positioned at the larger side of the b-CD
cavity. The well-known MM? method was used with the
`
Polak–Ribiere conjugate gradient to minimize the energy
Fig. 13 Molecular modeling of Vitamin B10—b-cyclodextrin inclu-
sion compound
of the structures of p-amino benzoic acid molecule and
b-CD jointly until a RMS gradient lower than 0.015 kcal/
˚
mol/A was obtained. Details of the algorithms are given
elsewhere [19]. Molecular modeling by molecular mechan-
ics established the geometry of the inclusion compound,
see Fig. 13, in agreement with the experimental (2D
NMR), PM3 and AM1 molecular modeling data [21].
As a result of this process, it was established that whole
Vitamin B10 molecule is included inside CD cavity, with
COOH group oriented toward secondary rim, whereas the
NH2 group is oriented to the primary rim of b-cyclodextrin.
Conclusions
–
–
mas(C=O) and ds(NH2) FTIR vibrational modes changes
certify the inclusion compound formation.
For kn and fd inclusion compounds products a mixture
of amorphous and crystalline phases with very small
crystallites were obtained, whereas a new quite crys-
talline inclusion compound was obtained by co product.
The decreasing of dehydration endothermic peak of
cyclodextrin and disappearance of the melting peak of
the Vitamin B10 in the DSC thermograms certify the
inclusion compounds formation.
–
–
–
The chemical shifts of the Vitamin B10 and cyclodex-
trin protons demonstrate the both the formation of an
inclusion complex between Vitamin B10 and b-cyclo-
dextrin with an 1:1 stoichiometry.
2D NMR experiments showed that Vitamin B10 is
included with the carboxylic group inside b-CD cavity
near to its narrower rim.
Fig. 12 Benesi–Hildebrand plot for the complexation of Vitamin
B10 with b-CD
123

182
–
J Incl Phenom Macrocycl Chem (2010) 68:175–182
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–
Based on molecular modeling using MM? approxi-
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Acknowledgments The authors kindly acknowledge the financial
support from the Romanian Ministry of Research and Education, Core
Project PN-09-44 02 01. Special thanks are due to Drs. M. Bogdan
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