Báo cáo khoa học: Interaction of gymnemic acid with cyclodextrins analyzed by isothermal titration calorimetry, NMR and dynamic light scattering doc

Interaction of gymnemic acid with cyclodextrins analyzed
by isothermal titration calorimetry, NMR and dynamic
light scattering
Yusuke Izutani
1
, Kenji Kanaori
2
, Toshiaki Imoto
3
and Masayuki Oda
1
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 glucose 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:
(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 10
5
)10
6
m
)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.
6154 FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS
Cyclodextrins (CDs) are cyclic oligosaccharides com-
posed of a-(1fi 4)-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 (K
a
), 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.
Results
ITC analysis
Figure 2 shows typical ITC profiles at 25 °C for the
interactions between GA and CDs. Exothermic heat
was gradually decreased after each injection of GA
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 integrated values. The corrected 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 25 °C 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 P
i
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 35 °C (Table 2). Through-
out the temperature range analyzed, binding is

Fig. 1. Chemical structure of GA.
Y. Izutani et al. Interaction of gymnemic acid with cyclodextrins
FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS 6155
accompanied 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 (DC
p
) ¼ )0.14 kcalÆ
mol
)1
Æ°C
)1
, assuming that DC
p
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
1
H NMR signals of GA alone were much broader in
D
2
O than those reported in pyridine-d
5
containing
a few drops of D

2
O [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.
pH n
a
K
a
(· 10
5
M
)1
) DG (kcalÆmol
)1

) DH
b
(kcalÆmol
)1
) TDS (kcalÆmol
)1
)
GA injection into c-CD
4.5
c
1.01 3.3 ± 0.4 )7.5 )4.5 3.0
5.5
c
1.01 4.5 ± 0.5 )7.7 )4.5 3.2
7.4
d
1.02 5.5 ± 0.5 )7.8 )4.4 3.4
7.4
e
1.02 5.5 ± 0.5 )7.8 )4.2 3.6
9.5
f
1.03 5.2 ± 0.5 )7.8 )4.7 3.1
c-CD injection into GA
7.4
d
1.01 4.3 ± 0.5 )7.7 )4.5 3.2
a
The n-value represents binding stoichiometry of GA to c-CD. The fitting error was < 1%.
b

The fitting error was < 2%.
c
In 50 mM sodium
acetate buffer,
d
in 50 mM Tris ⁄ HCl buffer,
e
in 50 mM phosphate buffer,
f
in 50 mM glycine ⁄ NaOH buffer.
Interaction of gymnemic acid with cyclodextrins Y. Izutani et al.
6156 FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS
(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 H
2
O 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.
Table 2. Thermodynamic parameters of the interaction between GA and c-CD at pH 7.4.
Temperature (°C) n
a
K
a

(· 10
5
M
)1
) DG (kcalÆmol
)1
) DH
b
(kcalÆmol
)1
) TDS (kcalÆmol
)1
)
20.2 1.03 5.7 ± 0.6 )7.7 )3.7 4.0
25.1 1.02 5.5 ± 0.5 )7.8 )4.4 3.4
30.1 1.01 5.1 ± 0.5 )7.9 )5.3 2.6
35.1 1.03 5.1 ± 0.4 )8.1 )5.8 2.3
All measurements were performed for GA injection into c-CD in 50 m
M Tris ⁄ HCl buffer (pH 7.4).
a
The n-value represents binding stoichiom-
etry of GA to c-CD. The fitting error was < 1%.
b
The fitting error was < 1%.
Fig. 3.
1
H 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.
Y. Izutani et al. Interaction of gymnemic acid with cyclodextrins
FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS 6157
Discussion
We 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 $ 10
5
)10
6
m
)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
DC
p
value, )0.14 kcalÆmol
)1
Æ°C
)1
, is large and negat-
ive, which is in the largest range among those of other
interactions with CDs [15]. Because negative values for
DC
p
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 DC
p
value, )0.14 kcalÆmol
)1
Æ°C
)1
, would be larger
than predicted by the correlation between DC
p
and
accessible surface area upon binding [24,25], suggesting

that other effects such as salt dehydration might con-
tribute to the DC
p
value 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 mM GA. (B) Relative frequency of
39 m
M c-CD. DLS of c-CD at low concentration such as 3.1 mM was difficult to detect. (C) Relative frequency of 3.1 mM GA in the presence
of equimolar concentration of c-CD. Relative frequency of molecule number was shown against the logarithm of molecule diameter.
Interaction of gymnemic acid with cyclodextrins Y. Izutani et al.
6158 FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS
intra- and intermolecular NOE peaks were observed
for the complex (unpublished results). Together with
the NMR results, an ITC study indicated that the

aglycone moiety of GA penetrates into the c-CD
pocket. When the complex structure has been deter-
mined at atomic resolution, it will be interesting to
analyze the contribution of each interaction, such as
hydrogen 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), K
a
and DH, were
obtained from the fitted curve. The values of DG and DS
were calculated from the equation,
DG ¼ÀRT lnK
a
¼ DH À T DS ð1Þ
where R is the gas constant, and T is absolute temperature.
The heat capacity change, DC
p
, was calculated from the
linear fitting to the D H values measured at various
temperatures, assuming that DC
p
is constant within the
experimental temperatures,
DC
p
¼ðDH=DTÞð2Þ
NMR measurements
For the acquisition of NMR spectra, GA was dissolved in

D
2
O 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.
1
H NMR spectra were
measured on a Bruker ARX-500 at 30 °C.
1
H chemical
shifts were referred to internal sodium 3-(trimethyl-
silyl)propionate-2,2,3,3-d
4
.
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 H
2
Oat25°C. 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.
References
1 Warren RM & Pfaffmann C (1959) Suppression of

sweet sensitivity by potassium gymnemate. J Appl Phy-
siol 14, 40–42.
2 Kurihara Y (1969) Antisweet activity of gymnemic acid
A1 and its derivatives. Life Sci 8, 537–543.
3 Yoshioka S (1986) Inhibitory effects of gymnemic acid
and an extract from the leaves of Zyzyphus jujuba on
glucose absorption in the rat small intestine. J Yonago
Med Assoc 37, 142–145 (in Japanese).
4 Hirata S, Abe T & Imoto T (1992) Effects of crude gym-
nemic acid on the oral glucose tolerance test in human
beings. J Yonago Med Assoc 43, 392–396 (in Japanese).
5 Nakamura Y, Tsumura Y, Tonogai Y & Shibata T
(1999) Fecal steroid excretion is increased in rats by oral
administration of gymnemic acids contained in Gym-
nema sylvestre leaves. J Nutr 129, 1214–1222.
6 Porchezhian E & Dobriyal RM (2003) An overview on
the advances of Gymnema sylvestre: chemistry, pharma-
cology and patents. Pharmazie 58, 5–12.
7 Suttisri R, Lee IS & Kinghorn AD (1995) Plant-derived
triterpenoid sweetness inhibitors. J Ethnopharmacol 47,
9–26.
Y. Izutani et al. Interaction of gymnemic acid with cyclodextrins
FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS 6159
8 Jiang P, Cui M, Zhao B, Liu Z, Snyder LA, Benard
LM, Osman R, Margolskee RF & Max M (2005) Lacti-
sole interacts with the transmembrane domains of
human T1R3 to inhibit sweet taste. J Biol Chem 280,
15238–15246.
9 Sugita D, Inoue R & Kurihara Y (1998) Sweet and
sweetness-inducing activities of new triterpene glyco-

sides, strogins. Chem Senses 23, 93–97.
10 Nelson G, Hoon MA, Chandrashekar J, Zhang Y,
Ryba NJ & Zuker CS (2001) Mammalian sweet taste
receptors. Cell 106, 381–390.
11 Zhao GQ, Zhang Y, Hoon MA, Chandrashekar J,
Erlenbach I, Ryba NJ & Zuker CS (2003) The receptors
for mammalian sweet and umami taste. Cell 115,
255–266.
12 Nagaoka T, Hane H, Yamashita H & Kensho I (1990)
Taste improvement of extracts from the leaves of Gym-
nema sylvestre. Seitou Gijutsu Kenkyukai-Shi 38, 61–70
(in Japanese).
13 Imoto T (1990) Sugar discrimination and plant-origin-
ated glycosides, gymnemic acids. Seibutsu Butsuri 30,
146–150 (in Japanese).
14 Szejtli J (1998) Introduction and general overview of
cyclodextrin chemistry. Chem Rev 98, 1743–1753.
15 Rekharsky MV & Inoue Y (1998) Complexation
thermodynamics of cyclodextrins. Chem Rev 98, 1875–
1918.
16 Wimmer R, Aachmann FL, Larsen KL & Petersen SB
(2002) NMR diffusion as a novel tool for measuring the
association constant between cyclodextrin and guest
molecules. Carbohydr Res 337, 841–849.
17 Davis ME & Brewster ME (2004) Cyclodextrin-based
pharmaceutics: past, present and future. Nat Rev Drug
Discov 3, 1023–1035.
18 Wiseman T, Williston S, Brandts JF & Lin L-N (1989)
Rapid measurements of binding constants and heats of
binding using a new titration calorimeter. Anal Biochem

179, 131–137.
19 Fukada H & Takahashi K (1998) Enthalpy and heat
capacity changes for the proton dissociation of various
buffer components in 0.1 m potassium chloride. Proteins
33, 159–166.
20 Liu HM, Kikuchi F & Tsuda Y (1992) Isolation and
structure elucidation of gymnemic acids, antisweet prin-
ciples of Gymnema sylvestre. Chem Pharm Bull 40,
1366–1375.
21 Chen W, Chang CE & Gilson MK (2004) Calculation
of cyclodextrin binding affinities: energy, entropy, and
implications for drug design. Biophys J 87, 3035–3049.
22 Harries D, Rau DC & Parsegian VA (2005) Solutes
probe hydration in specific association of cyclodextrin
and adamantane. J Am Chem Soc 127, 2184–2190.
23 Sturtevant JM (1977) Heat capacity and entropy chan-
ges in processes involving proteins. Proc Natl Acad Sci
USA 74, 2236–2240.
24 Murphy KP & Freire E (1992) Thermodynamics of
structural stability and cooperative folding behavior in
proteins. Adv Protein Chem 43, 313–361.
25 Spolar RS, Livingstone JR & Record MT Jr (1992) Use
of liquid hydrocarbon and amide transfer data to esti-
mate contributions to thermodynamic functions of pro-
tein folding from the removal of nonpolar and polar
surface from water. Biochemistry 31, 3947–3955.
26 Oda M, Furukawa K, Ogata K, Sarai A & Nakamura
H (1998) Thermodynamics of specific and non-specific
DNA binding by the c-Myb DNA-binding domain.
J Mol Biol 276, 571–590.

27 Oda M & Azuma T (2000) Reevaluation of stoichio-
metry and affinity ⁄ avidity in interactions between anti-
hapten antibodies and mono- or multi-valent antigens.
Mol Immunol 37, 1111–1122.
28 Schneider HJ, Hacket F, Rudiger V & Ikeda H (1998)
NMR studies of cyclodextrins and cyclodextrin
complexes. Chem Rev 98, 1755–1786.
29 Izutani Y, Murai T, Imoto T, Ohnishi M, Oda M &
Ishijima S (2005) Gymnemic acids inhibit rabbit glycer-
aldehyde-3-phosphate dehydrogenase and induce a
smearing of its electrophoretic band and dephosphoryla-
tion. FEBS Lett 579, 4333–4336.
Interaction of gymnemic acid with cyclodextrins Y. Izutani et al.
6160 FEBS Journal 272 (2005) 6154–6160 ª 2005 FEBS