These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of c-CD.. In addition, NMR measurements sh
Trang 1by isothermal titration calorimetry, NMR and dynamic
light scattering
Yusuke Izutani1, Kenji Kanaori2, Toshiaki Imoto3and Masayuki Oda1
1 Graduate School of Agriculture, Kyoto Prefectural University, Japan
2 Department of Applied Biology, Kyoto Institute of Technology, Japan
3 Faculty of Medicine, Tottori University, Yonago, Japan
Gymnemic acid (GA), a saponin of triterpene
glyco-side is contained in leaves of Gymnema sylvestre, which
is native to India, and has various physiological effects
such as an antisweet taste, inhibition of intestinal
glu-cose absorption, and lowering of plasma gluglu-cose and
insulin levels [1–6] As shown in Fig 1, GA is not a
pure entity, but is composed of several types of
homo-logues [7] Regarding the antisweet effect in humans,
when around 1 mm of partially purified GA in water is
tasted beforehand, the ability to taste anything sweet is
abolished for 30–60 min [2] Although it is not clear
how GA acts as an inhibitor, it is considered that GA
binds to the sweet taste receptor, similar to another
taste antagonist, lactisole [8] It should also be noted
that strogin, whose structure resembles that of GA,
has sweet and sweetness-inducing activity, and seems
to bind to the same site on the receptor [9] The sweet-taste receptor was recently identified to be a hetero-meric dimer of G-protein-coupled receptors (T1R2 and T1R3) expressed in subsets of taste receptor cells on the tongue and palate [10,11]
Interestingly, the antisweet taste effect of GA has been known to be immediately diminished by rinsing the tongue with c-cyclodextrin (c-CD) solution after
GA has been held in the mouth The restorative effect
of c-CD on the suppressed sweet taste by the extract of
G sylvestre was first reported by Nagaoka et al [12] and the same effect has been observed in case of
GA [13] Similarly, the sweet-inducing effect of strogin
is also diminished by application of c-CD [9]
Keywords
aggregation; cyclodextrin; gymnemic acid;
molecular interaction; thermodynamics
Correspondence
M Oda, Graduate School of Agriculture,
Kyoto Prefectural University, 1-5, Hangi-cho,
Shimogamo, Sakyo-ku, Kyoto 606-8522,
Japan
Fax: +81 75 703 5673
Tel: +81 75 703 5673
E-mail: oda@kpu.ac.jp
(Received 30 August 2005, revised 6
October 2005, accepted 11 October 2005)
doi:10.1111/j.1742-4658.2005.05014.x
The physiological phenomenon that the antisweet taste effect of gymnemic acid (GA) is diminished by application of c-cyclodextrin (c-CD) to the mouth was evaluated at the molecular level using isothermal titration calorimetry, NMR and dynamic light scattering These analyses showed that GA specifically binds to c-CD Thermodynamic analysis using isother-mal titration calorimetry revealed that the association constant of GA and c-CD is 105)106m)1 with favorable enthalpy and entropy changes The heat capacity change was negative and large, despite the change in access-ible surface area upon binding being small These thermodynamics indicate that the binding is dominated by hydrophobic interactions, which is in agreement with inclusion complex formation of c-CD In addition, NMR measurements showed that in solution the spectra of GA are broad and sharpened by the addition of c-CD, indicating that unbound GA is in a water-soluble aggregate that is dispersed when it forms a complex with c-CD Dynamic light scattering showed that the average diameter of unbound GA is > 30 nm and that of GA and c-CD complex is 2.2 nm, similar to unbound c-CD, supporting the aggregate property of GA and the inclusion complexation of GA by c-CD
Abbreviations
CD, cyclodextrin; DLS, dynamic light scattering; GA, gymnemic acid; ITC, isothermal titration calorimetry.
Trang 2Cyclodextrins (CDs) are cyclic oligosaccharides
com-posed of a-(1fi4)-linked a-d-glucosyl unit, in which the
most common and studied CDs are a-, b- and c-CDs,
consisting of 6-, 7- and 8-glucosyl units, respectively
[14] CDs can be described as toroidal, hollow,
trun-cated cones with a hydrophilic surface and a
hydropho-bic pocket, which forms an inclusion complex with an
organic compound, known as a host–guest interaction
[15,16] The hydrophobic pocket diameters of a-, b- and
c-CDs are 4.7–5.3, 6.0–6.5 and 7.5–8.3 A˚, respectively
[14] The unique properties of CDs are utilized in many
applications such as the pharmaceutical, food and
chemical industries: solubility enhancement,
stabiliza-tion of labile drugs, control of volatility and
sublimat-ion, physical isolation of incompatible compounds,
long-term protection of color, odor and flavor, and
sup-pression of hemolysis and the bitter tastes of drugs
[6,14,17] Taking into account the size of the
hydropho-bic pockets of CDs and the chemical structure of GA
(Fig 1), it can be speculated that c-CD forms an
inclu-sion complex with GA, diminishing the antisweet effect
of GA, although there has been no information about
their specific molecular interaction to date
In this study, we analyzed the interactions between
GA and CDs using isothermal titration calorimetry
(ITC), NMR and dynamic light scattering (DLS) ITC
measurements provide thermodynamic parameters, not
only the binding affinity (Ka), but also the enthalpy
change (DH) and entropy change (DS) [18] The
bind-ing experiments of GA to a-CD, b-CD and c-CD
showed that GA specifically interacts with c-CD To
explore the recognition mechanism between GA and
c–CD, the interaction was further analyzed under
dif-ferent conditions of pH, buffer and temperature In
addition, NMR and DLS analyses demonstrated the
characteristic properties of GA Our results of the
molecular interaction between GA and c-CD can be
correlated with the physiological phenomenon of sweet
taste modification
into c-CD, whereas only heat of dilution was observed after each injection of GA into the experimental buffer and a-CD (Fig 2A–C) When GA was injected into b-CD, small exothermic and endothermic heats were gradually titrated (Fig 2D) Although it is difficult to determine thermodynamics for the interaction between
GA and b-CD, owing to the small heats, the binding affinity of b-CD is much lower than that of c-CD des-cribed below These results clearly indicate that GA specifically binds to c-CD Assuming formation of the inclusion complex for these interactions, it could be deduced that the cavity size of c-CD is suitable for
GA binding, but those of a-CD and b-CD are too small In order to determine the binding site of GA, binding of glucuronic acid to c-CD was also analyzed, although that of gymnemagenin could not be examined due to its low solubility Because only heat of dilution was observed for the interaction between glucuronic acid and c-CD (data not shown), the aglycone portion
of GA should penetrate into the hydrophobic pocket
of c-CD
To determine the thermodynamic parameters, the area of each exothermic peak as observed in Fig 2B was integrated, and the heat of dilution was subtrac-ted from the integrasubtrac-ted values The correcsubtrac-ted heat was divided by the number of moles of GA injected, which were calibrated in consideration with the pur-ity of GA, and the resulting values were plotted as a function of the molar ratio The resultant data were best-fit according to a model for one binding site, using a nonlinear least-squares method (Fig 2E) The thermodynamic parameters at 25C are summarized
in Table 1 Similar thermodynamic parameters at dif-ferent pH values between 4.5 and 9.5 indicate that the chemical structures of both GA and c–CD and their interactions are little perturbed in this pH range The similar DH values at pH 7.4 in Tris⁄ HCl and Pi indicate that there is little effect of buffer ionization on complex formation [19] The binding stoichiometry of GA to c-CD shows that the com-plex forms in an equimolar ratio of respective mole-cules
ITC measurements of the interaction of GA and c-CD were further performed at four different temper-atures, ranging from 20 to 35C (Table 2) Through-out the temperature range analyzed, binding is
Fig 1 Chemical structure of GA.
Trang 3accompanied by favorable changes in enthalpy and
entropy, and shows strong temperature dependence for
both DH and TDS, which compensate each other to
make the Gibbs free energy change (DG) almost
insen-sitive to temperature The temperature dependence of
DH yields a heat capacity change (DCp)¼)0.14 kcalÆ
mol)1ÆC)1, assuming that DCp is constant within the
experimental temperatures
NMR analysis NMR methods were applied for the analysis of inter-actions between GA and CDs Line widths of
1H NMR signals of GA alone were much broader in
D2O than those reported in pyridine-d5 containing
a few drops of D2O [20], and those broad signals were unchanged by the addition of a- and b-CDs
Fig 2 Typical ITC profiles of the GA binding
to CDs A 2.2 m M solution of GA was injec-ted 16 times in increments of 10 lL into the experimental buffer (A), and a 0.1 m M solu-tion of c-CD (B), a-CD (C), and b-CD (D) at
25 C Titrations were performed over 10 s
at intervals of 180 s All samples were in
50 m M Tris ⁄ HCl buffer (pH 7.4) (E) The data points were obtained by integration of the peaks in (B), corrected for the dilution heat (A), and plotted against the molar ratio (GA ⁄ c-CD) The data were fitted using a nonlinear least-squares method.
Table 1 Thermodynamic parameters of the interaction between GA and c-CD at 25 C.
M )1) DG (kcalÆmol)1) DHb (kcalÆmol)1) TDS (kcalÆmol)1)
GA injection into c-CD
c-CD injection into GA
a
The n-value represents binding stoichiometry of GA to c-CD The fitting error was < 1%.bThe fitting error was < 2%.cIn 50 m M sodium acetate buffer, d in 50 m M Tris ⁄ HCl buffer, e in 50 m M phosphate buffer, f in 50 m M glycine ⁄ NaOH buffer.
Trang 4(Fig 3A) Because no precipitation was observed in these NMR samples, GA, at least above the concen-tration of 2.0 mm, would form a water-soluble aggre-gate In the presence of c-CD, however, the GA signals became sharp (Fig 3A) These results indicate that the aggregated GA in the aqueous solution is dispersed by the addition of c-CD but not a- or b-CDs The higher dispersion effect of c-CD is poss-ibly related to its high affinity for GA, as shown in the ITC experiments Thus, the concentration depend-ence of c-CD on the spectral change of GA was examined As the concentration of c-CD was increased the line width of GA became sharper, and above the equimolar c-CD to GA, the spectra were unchanged (Fig 3B) This is in accordance with the ITC results that c-CD and GA form a 1 : 1 complex Sharp singlet signals were observed around 1 p.p.m
in the complex of GA and c-CD, probably origin-ating from the methyl groups of the genin moiety [20] Taking the thermodynamic parameters into con-sideration, the NMR results indicate the specific bind-ing of GA to the pocket of c-CD occurs in the aqueous solution, accompanying dispersion of the self-association of GA
DLS analysis
In order to further analyze the aggregated property
of GA and the effects of c-CD, size distribution of
GA in the absence or presence of c-CD in H2O was analyzed by DLS (Fig 4) Two distributions were observed for a 3.1 mm solution of GA, in which the average radii are 37.1 ± 3.5 and 125.2 ± 34.0 nm, respectively (Fig 4A) These values are much larger than predicted from the chemical structure of GA, indicating that GA in solution is in the form of a water-soluble aggregate, which is in accordance with the NMR results The addition of c-CD changed the distribution to much smaller size, 2.2 ± 0.4 nm, which is similar to the size of c-CD itself (Fig 4B,C), supporting that the aggregate of GA is dispersed when it forms an inclusion complex with c-CD
All measurements were performed for GA injection into c-CD in 50 m M Tris ⁄ HCl buffer (pH 7.4) The n-value represents binding stoichiom-etry of GA to c-CD The fitting error was < 1%.bThe fitting error was < 1%.
Fig 3.1H NMR spectra (500 MHz) of GA in the absence or
pres-ence of CDs (A) NMR spectra of 2.0 m M GA in the presence of
2.0 m M a-CD, b-CD, and c-CD (B) The concentration dependence
of c-CD on NMR spectra of 2.0 m M GA The ratios of c-CD to GA
are indicated.
Trang 5We analyzed the interaction between GA and CDs at
a molecular level using ITC, NMR and DLS These
analyses showed that GA specifically binds to c-CD
This is in good agreement with the physiological
phe-nomenon in humans, in which the antisweet effect of
GA is diminished by the addition of c-CD to the
ton-gue The binding stoichiometry determined by ITC
and NMR revealed that GA forms a complex with
c-CD in the ratio of 1 : 1 The DLS results that the
molecular size of c-CD and GA complex is similar to
that of unbound c-CD indicate an inclusion
complexa-tion, that is, GA would fit into the hydrophobic
pocket of c-CD Considering that the GA used in this
study is a mixture of homologues [7], each homologue
of GA would interact with c-CD in similar manner
This is also supported by the NMR results that the
broad GA signals were sharpened by the addition of
c-CD Presumably because the small pockets of a-CD
and b-CD are not able to form stable complexes with
GA, the binding of a-CD to GA is not observed and
that of b-CD is much weaker than that of c-CD The
difference in binding affinity toward guest molecules
depending on the size of CDs has also been reported
for other host–guest interactions [21]
The binding affinities of GA to c-CD were shown to
be 105)106m)1 under physiological conditions The
binding strength may explain the physiological
phe-nomenon that the sweet-suppressing activity of GA is
immediately diminished by application of 5 mgÆmL)1
(3.9 mm) c-CD to the mouth after 3 mgÆmL)1
(3.7 mm) GA has been held in the mouth [13] The
specific binding of c-CD would cause dissociation of
GA from the sweet taste receptor, resulting in recovery
of the sweet taste Although neither the concentration
of sweet taste receptors on the tongue nor the binding
affinity of GA to the receptor has been determined,
the need for c-CD in the mm range would correlate
with the binding affinity between GA and the receptor
The thermodynamic parameters obtained are in the range of those of other interactions with c-CD [15] The interaction of GA with c-CD is accompanied by favorable DS values together with favorable DH values (Tables 1 and 2) Because a large decrease in configu-rational entropy has been reported for CD complexa-tion, a dehydration effect upon binding would contribute to the favorable DS value observed with the interaction of GA and c-CD [21,22] In addition, the
DCp value, )0.14 kcalÆmol)1ÆC)1, is large and negat-ive, which is in the largest range among those of other interactions with CDs [15] Because negative values for
DCp are believed to arise from hydrophobic interac-tions, which release the structured water surrounding the nonpolar groups on the surface of uncomplexed protein [23], the GA and c-CD association would be dominated by hydrophobic interactions, which contrib-ute to the favorable DS observed in this association The DCpvalue,)0.14 kcalÆmol)1ÆC)1, would be larger than predicted by the correlation between DCp and accessible surface area upon binding [24,25], suggesting that other effects such as salt dehydration might con-tribute to the DCpvalue determined for the interaction
of GA and c-CD [22,26]
Analyses using NMR and DLS showed the aggrega-ted property of GA Aggregation should be due to the hydrophobic property of GA, which is also the main driving force for the interaction with c-CD as described above For the durability of antisweet effect of GA on
a human tongue, this water-soluble aggregation might
be important for its function of sweet antagonism It is hypothesized that the GA molecules in the aggregated form can simultaneously bind to several sweet taste receptors This may increase the durability of antisweet effect, particular because of the slow dissociation rate, which is also seen in antigen–antibody interactions as the avidity effect [27] The dispersion of GA aggregate
by c-CD may help to dissociate from the receptor
In order to elucidate the complex of GA and c-CD
in detail, NMR assignment is under way [28] Several
Fig 4 Particle size distribution of GA in the absence or presence of c-CD (A) Relative frequency of 3.1 m M GA (B) Relative frequency of
39 m M c-CD DLS of c-CD at low concentration such as 3.1 m M was difficult to detect (C) Relative frequency of 3.1 m M GA in the presence
of equimolar concentration of c-CD Relative frequency of molecule number was shown against the logarithm of molecule diameter.
Trang 6hydrogen bond and hydrophobic contacts and
hydra-tion upon binding on thermodynamics and its
correla-tion with structure These analyses may help us to
generally understand the molecular recognition
mech-anism of GA, and to design rationally new materials
to control the sense of taste
Experimental procedures
Materials
Thirty percent ethanol extract of G sylvestre was kindly
provided by Dai-Nippon Meiji Sugar Co., Ltd (Tokyo,
Japan) GA was purified from the extract as described
pre-viously [29] The GA sample obtained was a mixture of
homologues and its purity was 70% Because very
intri-cate and tedious steps are required to purify each
homo-logue, the mixture of homologues was used as GA in this
study, similar to most of other investigations that analyze
the functions of GA Gymnemagenin and Diaion HP20
were purchased from Maruzen Pharmaceuticals Co., Ltd
(Onomichi, Japan) and Mitsubishi Chemical Co., Ltd
(Tokyo, Japan), respectively All other reagents were
pur-chased from Nacalai Tesque, Inc (Kyoto, Japan)
ITC measurements
MCS-ITC (Microcal, Northampton, USA) was used for
thermodynamic analysis of the interaction between GA and
CDs All samples dissolved in buffers were filtrated through
a 0.45 lm filter and degassed before the titrations, using
the equipment provided with the instrument GA or CD
solution (2.2 mm) was titrated into CD or GA solution
(0.1 mm) using a 250-lL syringe Each titration consisted
of an initial injection (2.5 lL) followed by 15 main
injec-tions (10 lL)
Measurement data were analyzed by microcal origin
version 2.9 The resultant data was best-fit, according to a
model for one binding site using a nonlinear least-squares
method The binding stoichiometry (n), Ka and DH, were
obtained from the fitted curve The values of DG and DS
were calculated from the equation,
DG¼ RT ln Ka¼ DH TDS ð1Þ
where R is the gas constant, and T is absolute temperature
The heat capacity change, DCp, was calculated from the
linear fitting to the DH values measured at various
D2O at a concentration of 2.0 mm The pH of the solution was 4.6 (meter reading of glass electrode without correction
to pD) where the binding manner between GA and c-CD
is identical to that at neutral pH 1H NMR spectra were measured on a Bruker ARX-500 at 30C 1
H chemical shifts were referred to internal sodium 3-(trimethyl-silyl)propionate-2,2,3,3-d4
DLS measurements
DLS-7000 (Otsuka Electronics Co., Ltd) was used to meas-ure DLS to estimate the diameters of GA, c-CD, and their mixture in H2O at 25C The sample was filtrated through
a 0.2 lm filter The measurement was performed using a laser beam of 488 nm at an angle of 90 The molecule number of diameter distribution was obtained by the histo-gram analysis
Acknowledgements
The authors thank Mr Shoichi Nakamura of Otsuka Electronics Co., Ltd for support of DLS measurements
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