Spectroscopic studies on the inclusion behavior between caffenic acid and γ-cyclodextrin

Article abstract of DOI:10.1007/s10847-011-9930-9

To investigate the inclusion ability of γ-cyclodextrin (γ-CD) for caffenic acid (CA). The conditions for the formation of inclusion complex and the binding constant between γ-CD and CA were determined by fluorescent and ultraviolet spectroscopic methods. The behavior of CA as a free radical scavenger before and after its inclusion was investigated. In addition, solid samples of the inclusion complex, prepared through the co-precipitation and grinding methods, were characterized via IR spectroscopy and differential scanning calorimetry. The inclusion complex was further characterized with ¹H NMR spectroscopy. By using fluorescent and ultraviolet spectroscopy, the conditions for the formation of inclusion complex between γ-CD and CA were optimized and the binding constant determined. It was observed that the guest molecule behaves as a better anti-oxidant after its inclusion into γ-CD.

Full text of DOI:10.1007/s10847-011-9930-9

J Incl Phenom Macrocycl Chem (2011) 71:25–34
DOI 10.1007/s10847-011-9930-9
REVIEW ARTICLE
Spectroscopic studies on the inclusion behavior between caffenic
acid and c-cyclodextrin
•
•
•
Wei Zhao Jianbin Chao Rui Du
Shuping Huang
Received: 16 July 2010 / Accepted: 20 January 2011 / Published online: 11 February 2011
Ó Springer Science+Business Media B.V. 2011
Abstract To investigate the inclusion ability of c-cyclo-
dextrin (c-CD) for caffenic acid (CA). The conditions for
the formation of inclusion complex and the binding con-
stant between c-CD and CA were determined by fluores-
cent and ultraviolet spectroscopic methods. The behavior
of CA as a free radical scavenger before and after its
inclusion was investigated. In addition, solid samples of the
inclusion complex, prepared through the co-precipitation
and grinding methods, were characterized via IR spec-
troscopy and differential scanning calorimetry. The inclu-
sion complex was further characterized with 1H NMR
spectroscopy. By using fluorescent and ultraviolet spec-
troscopy, the conditions for the formation of inclusion
complex between c-CD and CA were optimized and the
binding constant determined. It was observed that the guest
molecule behaves as a better anti-oxidant after its inclusion
into c-CD.
molecular recognition is the driving force for the formation
of supramolecular structures and also forms the basis for
the understanding of the selective binding between the host
and the guest molecules. In this respect, there exist three
types of the most attractive host molecules: cyclodextrins
(CDs), crown, and calixarenes. All three types of molecules
have been used as molecular models for biomolecule.
Therefore, the investigation of the interaction of these host
molecules with small organic molecules as well as the
physical chemical properties of these interactions has
become an important research area. Among them, CDs are
by far the most important host molecules.
As host molecules, CDs have many interesting and
useful properties [2]. With a lipophilic interior and a
hydrophilic exterior, CDs can form various inclusion
complexes with many organic and inorganic molecules. As
the microenvironment inside the CD cavity differs from
that of an aqueous solution, the guest molecule often dis-
plays rather different photochemical and photophysical
properties. CDs have been extensively used for the for-
mation of supramolecular complexes by selective binding
to their substrates (or guests) and research interests in this
area can be seen in various fields of chemistry and biology.
CDs are oligomers of glucose (Fig. 1a). Depending on their
size, CDs can be a, b, or c. The researches associated with
c-cyclodextrin (c-CD), which has eight glucose units, are
relatively fewer than those of its a and b analogues. On the
other hand, c-CD has a larger cavity and should in principle
form inclusion complexes with a bigger variety of guest
molecule [3].
Keywords Caffenic acid ꢀ Cyclodextrins ꢀ Inclusion
compounds
Introduction
Molecular recognition is among the most active research
areas in the chemistry of supramolecules [1]. Similar to the
‘‘key and lock’’ binding principle of biological molecules,
W. Zhao ꢀ J. Chao (&) ꢀ S. Huang
The Institute of Applied Chemistry of Shanxi University,
Taiyuan 030006, People’s Republic of China
e-mail: chao@sxu.edu.cn
CA, shown in Fig. 1b, is a naturally occurring phenyl-
propanoid and is found in many Chinese herbs. CA has
been shown to be possessed general antibiotic, antiviral,
and hemostatic properties. It is also found to be able to
increase the level of human leukocytes.
R. Du
Department of Engineering, Shanxi Coal Mining Administrators
College, Taiyuan 030006, People’s Republic of China
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J Incl Phenom Macrocycl Chem (2011) 71:25–34
Experimental section
water and shaken thoroughly, and ultrasonic treatment was
applied to the mixture at 20 1 °C for 30 min. On the one
hand, effect of the concentration of the CD was investi-
gated by fluorescence spectroscopy. On the other hand, the
temperature was measured by the UV absorption at 25, 30,
37, and 45 °C, respectively. Finally, pH effects on the
formation of the inclusion complex were investigated by
fluorescence spectroscopy, varying the pH from 3.05 to
8.96.
Reagents
CA was dissolved in water to prepare a 1.0 9 10-3 mol/L
solution, and c-CD were prepared for the solutions of
1 9 10-2 mol/L. All the other reagents were of analytical-
reagent grade and were used as received. 1,1-diphenyl-2-
picrylhydrazyl (DPPH) (Tokyo, Japan Chemical Industry
Co., Ltd.) was used in clearing free radicals experiment and
need prepared for the concentration with the same of CA
solutions. Phosphate buffer solutions of NaH2PO4/H3PO4
were used to control the pH = n (n = 3.05, 5.0, 6.5, and
8.96) of the working media. Doubly distilled water was used
throughout. All experiments were carried out at room
temperature.
Apparatus
The UV absorption experiments were carried out on a
UV-757CRT spectrophotometer (Shanghai Precision &
Scientific Instrument Co., Shanghai, China). Fluorescence
measurements were conducted on a Hitachi F-2500 FL
spectrofluorimeter (Tokyo, Japan) using an excitation
wavelength of 285 nm and emission wavelength of 445 nm.
Excitation and emission slit width were both set at 10 nm. IR
spectra were obtained with FT-1370 IR spectrometer using
KBr pellets. NMR spectra were recorded on a DRX-300
¨
spectrometer (Fallenden, Switzerland) using D2O as solvent.
Differential scanning calorimetry (DSC) analysis was car-
ried out on DSC-60 thermal analyzer (Shimadzu, Japan).
Procedure
Analysis of the conditions of inclusion complex formation
1 mL of the CA stock solution was transferred into a
10 mL volumetric flask, and then an appropriate volume of
1.0 9 10-2mol/L c-CD solution was added. The mixed
solution was diluted to 10 mL final volume with distilled
Fig. 2 The spetras of CA in the presence of various amounts of
c-CD. a Fluorescence spectra, b UV spectra
Fig. 1 Structure of CD and CA
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J Incl Phenom Macrocycl Chem (2011) 71:25–34
27
complex solution and 0.5 mL contrast solution. By this
way, the rate of clearing free radical could be obtained.
Preparation of CA/c-CD solid complex
Solid complex of CA with c-CD was prepared with the
co-precipitation method or the grinding method [5].CD was
made of saturated aqueous solution, then 40% ethanol
solution of caffeic acid was dropped slowly into it, stired
constantly for a long time and cooled gradually to room
temperature. The solid precipitation of inclusion complex
was filtered, washed with a small amount of 40% ethanol,
and then dry to stable inclusion complex.
NMR measurements
Fig. 3 The reciprocal plot (1/[CD] Vs. 1/[F-F0]) for the inclusion
complex between CA and c-CD
Equal volumes of c-CD and CA solutions, both at con-
centrations of 1.0 9 10-4 mol/L, were mixed until com-
plete reaction, and NMR measurements were taken and
contrasted with c-CD and CA by Bruker Avancev
DRX300 MHz. All the spectra were recorded in 99.96%
D2O at 298 1 °C.
DPPH radical scavenging test
Fig. 4 Effect of temperature
Clearing of free radicals was used to determine the effi-
ciency of CA. We also examined thermal and photolysis
stability of CA. The same concentration of c-CD and CA
solutions of identical concentration were mixed at 80 °C
and were kept for 2 h, then UV absorbance was measured
every half hour and residual rate was calculated.[4] The
results were expressed as percentage of DPPH radical
elimination calculated according to the following equation:
Fig. 5 Plots of 1/[CD]*1/[F-F0] at different pH values
Â
À
Áꢀ
Ã
AU ¼ 1 ꢁ Ai ꢁ Aj Ac ꢂ 100%
ð1Þ
Table 1 The thermodynamic data for the inclusion of caffenic acid
in c-CD
To alcohol: water = 9:1 as a reference solution, Ai, Ac, and
Aj were measured by using UV absorbance spectroscopy.
Where AU is the radical-scavenging activity, Ai was con-
sidered as the absorbance of the mixture of 3 mL DPPH
and 0.5 mL complex solution, Ac represented the absor-
bance of the mixture of 3 mL DPPH and 0.5 mL contrast
solution, and Aj was the absorbance of the mixture of 3 mL
T
K
DG
-9.8
DH
DS
298.2
303.2
310.2
318.2
52.4
-80.5
-80.5
-80.5
-80.5
-360.3
-360.3
-360.3
-360.3
113.8
208.5
84.3
-11.9
-13.8
-11.7
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Fig. 6 Ionisation of CA from
pH 3.05 to pH 8.96
Results and discussions
It is apparent from Fig. 2 that the maximum absorption
underwent a red shift, which is also indicative of the for-
mation of an inclusion complex between CA and c-CD.
Based on the good linear correlation revealed in Fig. 3, it
can be concluded that the inclusion complex has a 1:1
stoichiometry with the binding constant being 943.
Conditions for inclusion complex formation
Effect of the CD concentration
Figure 2 gives the fluorescent spectra (a) and UV spectra
(b) of CA in the presence of various amounts of c-CD. It
can been seen from these spectra, the intensity of both the
fluorescence and the UV absorption increases with increase
of the c-CD concentration, indicating that the inclusion of
CA into the cyclodextin has occurred and the concentration
of the cyclodextin has an influence in the inclusion. Spe-
cifically, the inclusion interaction between the host and the
guest molecules increases with the increase in the con-
centration of c-CD.
According to the modified Benesi–Hildebrand equation
[6]:
1=ðF ꢁ F0Þ¼1=ð½CDꢃkaÞþ1=a
or 1=ðA ꢁ A0Þ ¼ 1=ð½CDꢃkaÞ þ 1=a
ð2Þ
where F and F0 represent the fluorescent intensity of CA in
the presence and in the absence of c-CD, respectively; and
A and A0 represent the intensity of its UV absorption in the
presence and in the absence of c-CD, respectively; [CD] is
the total concentration of c-CD, whereas k and a are con-
stants. A plot of the reciprocals 1/[CD] versus 1/[F-F0]
gives a straight line as shown in Fig. 3, from which the
binding constant can be calculated by dividing its intercept
with its slope.
Table 2 The maxima of fluorescence and UV absorption of the
CA–c-CD complex
pH
K
UV absorption
kex (nm)
Fluorescence
kex (nm)
3.05
5.0
57.5
320
286
310
285
280
280
279
269
168.5
377.1
Fig. 7 DPPH removal rates (Figs 7a and b should be skipped and
replaced by a table). a A comparison on the DPPH removal rate
between CA, CGA, FA and their inclusion complexes b The DPPH
removal rate of CA and its inclusion complexes with different CDs
6.5
8.96
1430.0
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29
Fig. 8 IR spectra. a CA–c-CD inclusion complex prepared by co-precipitation, b CA–c-CD inclusion complex prepared by grinding, c c-CD, d CA
Effect of the temperature
respectively. Linear regression analysis on the
1/[CD]*1/[A-A0] plots gave the binding constants
at these temperatures. The results were shown in
Fig. 4.
The UV absorption of CA in the presence of different
levels of c-CD was measured at 25, 30, 37, and 45 °C,
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Fig. 8 continued
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31
DPPH removal rate
The change in Gibbs free energy at different tempera-
tures can be calculated from DG = 2RTlnKT. According to
the Van’t Hoff equation [7]:
Figure 7a shows the comparison of the anti-oxidizing
property for CA and its derivatives before and after being
included in c-CD. It can be seen from Fig. 7 that DPPH
removal rate for CA is larger than the removal rates of
chlorogenic acid (CGA) and ferulic acid (FA), that is, the
order of DPPH removal rate is CA [ CGA [ FA. It is also
apparent that the DPPH removal rate increases significantly
when the inclusion complexes are formed and follows the
same trend, CA [ CGA [ FA. These results are consistent
with what has been proposed by Min Zhu and Fangquan
Wang[11]. It shows that these drugs underwent some
changes in their properties, resulting in an improvement in
their anti-oxidizing ability. It may be caused by an
increased activity once CA is included in the cavity of the
CD.
lnKT ¼ ꢁDH=RT þ DS=R
AplotoflnKT versus 1/T followed by linear regression analysis
yielded the values of DH and DS [7], which were tabulated in
Table 1.
ð3Þ
The negative value of DG as shown in Table 1 is con-
sistant with the expectation that the formation of the
inclusion complex is an energetically favored and sponta-
neous process. It is also obvious from the negative DS that
the order of the system has decreased upon formation of the
complex. The process is driven by its favorable negative
enthalpy change that overcompensates the unfavorable
negative entropy change.
Figure (7b) shows the DPPH removal rates of CA and of
CA complexes with c-CD, hydroxypropyl-b-cyclodextrin
(HP-b-CD) and b-CD. Again, it can be seen that the
removal rate has increased after the formation of the
inclusion complex and the rate is higher for the HP-b-CD
complex than that of the c-CD complex, and the rate is
relatively small for the c-CD complex. Nevertheless, the
c-CD complex can still improve the activity and should be
of certain value to the study of inclusion compounds.
Effect of pH on the formation of the inclusion complex
We measured UV spectra of aqueous solutions of CA in the
presence of c-CD at different pH values. The maximum of
UV absorption varies with pH undergoing a blue shift with
increasing pH value. At when the pH of the solution
changes from 3.05 to 8.96, the maximum absorption
undergoes a blue shift. A possible reason for this could be
that as the pH increases, there is a higher degree of ioni-
zation for the carboxyl group of CA. The effect of cross-
conjugation results (Cross-conjugation is a special type of
conjugation in a molecule, when in a set of three p-i bonds
only two pi-bonds interact with each other by conjugation,
the third one is excluded from interaction) from structure of
CA that plays a major role in the formation of the inclusion
complex. The ionization of the carboxyl group may cause
an overall reduction in the dipole moment of the molecule
and bring about a diminishment of the conjugation effect,
ultimately leading to the observed blue shift of its UV
spectrum.
Figure 5 shows the 1/[CD]*1/[F-F0] plots measured at
different pH values. All of the plots displayed a good linear
relationship and indicated the formation of a 1:1 inclusion
complex. The binding constants can be obtained by linear
regression analysis on the plots and listed in Table 2. It can
be seen from the data in Table 2 that the binding constant
K of the CA–c-CDDE complex increases as the pH
increases. This trend is related to the structure of CA,
whose dissociation equilibration is shown in Fig. 6 [8].
As the pH value increases, CA will be ionized into
different anions and these species may form inclusion
complexes with CD more easily. However, CA may
undergo changes in its properties under alkaline conditions.
Therefore, the optimal acidity for the formation of inclu-
sion complex should be at neutral pH as Fig. 6 [9].
Fig. 9 DSC melting curves A CA, B c-CD, C CA–c-CD inclusion
complex prepared by grinding, D CA–c-CD inclusion complex
prepared by co-precipitation
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400–1,602 cm-1, but the intensity of the peaks at
1,645–1,602 cm-1 corresponding to the benzene ring sig-
nificantly decreased, indicating that both the benzene ring
and its OH groups are included in the cavity of c-CD and
both methods have been successful in preparing the
inclusion complex [10].
Characterization of the solid form of the CA–CD
complex
The IR spectra of CA, c-CD and the inclusion complex are
shown in Fig. 8.
Figure 8 shows IR spectrum, and It can be seen from
these spectra that there are significant changes in the peaks
at 2,500–3,450 cm-1 between CA itself and the inclusion
complexes prepared either with the co-precipitation
method or the grinding method, with the big peak at
3,411 cm-1 remaining identical to that of the c-CD; there
wasn’t any bigger change between c-CD and CA-c-CD at
DSC Characterization of the solid CD inclusion
complex
The results of the DSC study are shown in Fig. 9. It can be
seen that CA melts at around 225 °C as indicated by the
Fig. 10 1H NMR spectra of
CA, c-CD and the CA–c-CD
inclusion complex (a) and CA
moiety of the inclusion complex
(b) The spectrum of the CA
moiety is enlarged
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Table 3 Changes in the chemical shifts
1
(1) H chemical shifts of free and complexed c-CD
1H
dc-CD free (ppm)
dc-CD complexed (ppm)
Dd(c-CD complexed-c-CD free)
H1
H3
H6
H5
H2
H4
4.969
3.800
3.768
3.736
3.530
3.496
4.971
3.815
3.784
3.730
3.535
3.462
0.002
0.015
0.016
-0.006
0.005
0.034
1
(2) H chemical shifts of free and complexed CA
1H
DCA free (ppm)
DCA complexed (ppm)
Dd(CA complexed-CA free)
H4
H1
H2
H3
H5
7.100
6.907
6.807
6.674
6.100
7.042
6.649
6.611
5.725
5.674
-0.058
-0.258
-0.196
-0.949
-0.426
the CD. A possible structure for the inclusion complex is
proposed as in Fig. 11.
Conclusion
CA has been examined for its ability to form a complex
with c-CD. The results clearly demonstrated that c-CD can
form a stable complex with CA and therefore can be used
for the encapsulation/release of this potent antioxidant. The
DPPH scavenging ability of CA-c-CD inclusion complex is
larger as that of free CA. IR, DSC, and NMR spectroscopic
studies show that the aromatic ring and the ethylene side
chain of CA were deeply included inside the CD cavity.
This study would be helpful to promote the application of
caffenic acid.
Fig. 11 Proposed structure of the inclusion complex
exothermic peak at this temperature(Fig. 9A); for c-CD
itself (Fig. 9B), several melting peaks that vary in their
intensity appear between 270 and 320 °C; however, for the
inclusion complex (Fig. 9C and D), there is only one
endothermic peak at 320 °C. These results indicate that the
interaction between CA and c-CD is accompanied by the
appearance of a new phase that indicates the formation of
an inclusion complex [11, 12].
1H NMR characterization of the CD inclusion complex
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