The interactions of epigallocatechin-3-gallate with human

whole saliva and parotid saliva

Jiang-Wu Yao
a,
*, Chang-Jian Lin
b
, Guo-Yang Chen
a
, Feng Lin
a
, Tao Tao
c
a
Department of Restorative Dentistry and Biomaterials, Xiamen Stomatological Research Institute, Fujian Medical University,
2 DouXi Street, Xiamen, Fujian 361001, China
b
State Key Laboratory of Physical Chemistry of Solid Surfaces, Xiamen University, 422 South Siming Street, Xiamen, Fujian, China
c
Department of Biology, School of Life Sciences, Xiamen University, 422 South Siming Street, Xiamen, Fujian, China
1. Introduction
The biological effects of tea polyphenols are most often
attributedto tea catechins, whichare typical polyphenols such
as catechin, epicatechin, epicatechin gallate, epigallocatechin,
and epigallocatechin-3-gallate (EGCG).
13
Among these poly-
phenols, more than50%of the mass of this catechinmixture is
EGCG (Fig. 1).
2
The phenomenon of astringency in the oral
cavity is thought to be due to the interaction of salivary
proteins with tea polyphenols.
3,4
Therefore, EGCG is speculat-
ed to be the key tastant in green tea and to be a highly
astringent compound.
3
The association of polyphenol with proteins has been
largely studied in solutions.
5,6
The available methods used to
a r c hi v e s o f or a l b i o l og y 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8
a r t i c l e i n f o
Article history:
Accepted 24 April 2010
Keywords:
Saliva
EGCG
QCM-D
Quenching
Afnity
a b s t r a c t
Objective: The aim of the present study was to assess the null hypothesis that the astrin-
gency and loss of lubrication in the oral cavity are not related to the properties of the
epigallocatechin-3-gallate (EGCG) adlayer, the afnity and the entropy-drive of EGCG bind-
ing to saliva.
Methods: The mass, thickness, and viscoelasticity of the EGCG adlayer and the temperature-
dependence of EGCG adsorption onto saliva surfaces were determined by quartz crystal
microbalance with dissipation (QCM-D). The afnities of EGCG to human whole saliva (WS)
and to parotid saliva (PS) were carried out by QCM-D monitoring and uorescence quench-
ing.
Results: The stiffer and more compact EGCG adlayers were formed on saliva surfaces at
various concentrations of EGCG. The afnity for EGCGbinding to WS was higher than that to
PS. The precipitation of EGCG/saliva was temperature-dependent. The driving force of EGCG
binding to saliva is dominated by the hydrogen bond, the hydrophobic reaction, and the
entropy-drive, which were conrmed by the FTIR spectra and the measurement of temper-
ature-dependence, respectively.
Conclusion: The viscoelasticity of the EGCG adlayer, the afnity of EGCG to saliva, and the
priority of EGCG binding to hydrophobic proteins on the mucosa may account for the oral
astringency and loss of lubrication.
# 2010 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: +86 0592 2100928; fax: +86 0592 2139597.
E-mail address: dentyjw@126.com (J.-W. Yao).
Abbreviations: WS, whole saliva; PS, parotid saliva; EGCG, epigallocatechin-3-gallate; QCM-D, quartz crystal microbalance with
dissipation; K
L
, Langmuir constant; M
m
, maximum amount adsorbed; K
f
, Freundlich constant; DF, shifts in frequency; DD, shifts in
energy dissipation; m, adsorbed mass; h, adsorbed thickness; m, elasticity; h, viscosity; k
q
, bimolecular quenching constant; [Q],
concentration of quencher; K
SV
, SternVolmer constant; K
D
, dynamic quenching constant; K
S
, static quenching constant.
avai l abl e at www. sci encedi r ect . com
journal homepage: http://www.elsevier.com/locate/aob
00039969/$ see front matter # 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.archoralbio.2010.04.005
analyze the binding of polyphenol with proteins are energy
and time-consuming, rather than in situ techniques. These
techniques, however, cannot provide real-time information of
the binding process.
3
Quartz crystal microbalance with
dissipation (QCM-D) monitoring is traditionally used in the
laboratories of analytical and electro-analytical chemistry; it
has recently become a powerful, in situ, and real-time
technique used to study various biological interactions
between polyphenols and proteins at the liquid/solid inter-
faces.
7
Fluorescence quenching has also been widely used for
monitoring the binding of polyphenols to proteins because of
its sensitivity, accuracy, rapidity and ease of application.
8
Due to both the complexity of experimental design and the
lack of highly sensitive techniques, little is known about the
molecular basis of the sensation of astringency and lubrica-
tion in the oral cavity inuenced by the interactions between
polyphenols and proteins and, particularly, the mixed salivary
proteins. Therefore, the aim of the present study was to
evaluate the null hypothesis that the astringency and loss of
lubricationinthe oral cavity are not related to the properties of
the EGCG adlayer, the afnity and the entropically driven
characteristic of EGCG binding to salivary proteins on the oral
mucosa. Therefore, the rst objective was to use the QCM-D
technique to study EGCG, a typical tea polyphenol, absorption
onto gold-coated quartz crystal surfaces. Human whole saliva
(WS) and parotid saliva (PS) were chemically immobilized onto
the surfaces using thiolate self-assembled monolayers. The
absorbed mass, thickness, viscoelasticity, and the entropically
driven component of EGCGadsorption on saliva surfaces were
then determined by QCM-D. The afnities of EGCG binding to
WS and PS were analyzed by QCM-Dand quenching of salivary
protein intrinsic uorescence.
2. Materials and methods
2.1. Materials
99% EGCG and 11-mercaptoundecanoic acid (11-MUA) were
purchased from Sigma Chemical Co. N-hydroxysuccinimide
(NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC) were from Thermo (USA). Acetic acid,
sodium acetate, ammonium hydroxide (NH
4
OH), hydrogen
peroxide (H
2
O
2
), sodium chloride (NaCl), and absolute ethanol
(Aldrich, USA) were all used as received. The buffers used were
0.005 M phosphate buffer at pH 7.0 and 0.01 M sodium acetate
buffers at pH 4.9.
2.2. Saliva collection and protein characteristics
Stimulated human whole saliva and parotid saliva were
collected from all 10 volunteers (4 males, 2450-year old; 6
females, 2154-year old; all of them are non-smokers) in the
morning before noon. The collection of saliva samples began
10 min after the subjects had brushed their teeth for 1 min,
without toothpaste, to avoid any effects of food and drink. For
the collection of saliva samples, the subjects were instructed to
bite a piece of cotton wool (Salivette; Sarstedt Inc., Numbrecht,
Germany). Stimulatedwhole salivaproducedintherst minute
was discarded; thereafter, saliva was allowed to run from the
lower lip into pre-weighed tubes on ice to avoid foaming.
Human parotid saliva was collected directly fromthe Stensens
duct orice with the aid of a plastic suction cup (modied
Lashley cup). Immediately after collection, the saliva samples
were immediately stored on ice to prevent proteolytic degrada-
tion. Saliva was separatedfromthe cottonroll bycentrifugation
at 1200 g for 10 min. The ltrate was then snap-frozen at
40 8C, thawed, and centrifuged at 14,000 g for an additional
30 min to remove whole cells or their fragments. The super-
natants were pooled together (812 ml), divided into aliquots,
transferred to new tubes, and stored at 70 8C until needed.
9
The protein concentrations of WS and PS were determined
by the improved Bradford method.
10
A standard curve was
prepared using bovine serumalbumin (BSA) (Sigma). Linearity
was observed in the absorbance response at 595 nm to the
concentration of BSA (data not shown).
2.3. Preparation of saliva-modied quartz crystal surfaces
The immobilization of salivary proteins on the gold-coated
quartz crystal (fundamental frequency of 5 MHz, KVS, Finland)
surface was carried out as previously described
11
: the surfaces
of the gold electrodes were treated with piranha solution (98%
H
2
SO
4
+ 30% H
2
O
2
) for 5 min, and cleaned in a UV/ozone
(T10X10/OES-E UVOCS, USA) chamber for 10 min. After the
treatment, the crystals were rinsed thoroughly with pure
ethanol and ion exchanged water, and dried with nitrogen
gas (N
2
). When the cleaning process was nished, the crystal
was soakedin10 mM11-MUA/pure ethanol solutionat 60 8Cfor
at least 24 h. The excess 11-MUA on the surface of the sensor
was rinsed at least three times with pure ethanol and ion
exchangedwater, andthenthemodiedquartzcrystal surfaces
were dried with N
2
. A mixed solution containing 1:1 (v/v) of
100 mg/ml EDCand100 mg/ml NHSwas usedtoactivatethe 11-
MUA-coated quartz crystal surfaces for 1 h before the immobi-
lization of salivary proteins. A solution of 0.8 mg/ml salivary
protein in phosphate buffer (pH 7.0) was used to incubate the
activated surfaces at 25 8C for at least 24 h. Finally, the quartz
crystal surfaces were rinsed thoroughly by the phosphate
buffer, followed by distilled water, and dried with N
2
.
Fig. 1 The structure of EGCG.
a r c hi v e s o f or a l b i ol o gy 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 471
2.4. FTIR measurements
Infrared spectra of salivary protein-modied quartz crystal
surfaces before and after the adsorption of 12 mM EGCG were
collected with a Fourier transform infrared spectrometer
(FTIR, Thermo Nicolet 670, Madison, WI), using a pure gold
surface as the background. A Thermo Nicolet smart apertured
grazing angle (SAGA) accessory with a grazing angle of
incidence of 808 was used to collect reection absorption
infrared spectra. The resolution was set to 4 cm
1
, and 1024
scans were collected.
2.5. QCM-D measurements
The adsorption of EGCG to WS and to PS surfaces was studied
using a commercial QCM-D apparatus (QCM-Z500, KSV
Instruments, Finland). The temperature-controlled chamber
was initially lled with ion exchanged water at pH 7.0. After a
stable baseline was established, the EGCG solutions at
different concentrations were exposed to the salivary pro-
tein-modied crystal surfaces. At the same time, the adsorp-
tion was monitored as a function of time by recording the
shifts in frequency (DF) and energy dissipation (DD) simulta-
neously at the fundamental resonant frequency along with
the third, fth, and seventh overtones until a steady state of
adsorption was reached. The long-term stability of frequency
was within 1 Hz/15 min. The data analysis, calculations of
adsorbed mass (m), thickness (h), elasticity (m), and viscosity (h)
were performed by means of custom written QCM impedance
analysis software (KSV Instruments, version 3.11), at a
resolution of 2 s. Liquid ow through the QCM cell was
achieved using a QCM507 peristaltic pump and 2.5 ml syringe,
at a ow rate of 5 ml/min. Temperature-dependent QCM-D
measurements for the 12 mMEGCGsolutionadsorptiononWS
and PS surfaces were also carried out at various temperatures
at pH 7.0. The complete experiment was repeated three times.
When the adsorption equilibrium is established, the
Langmuir isotherm can be described by the equation
12
:
C
M

1
K
L
M
m

C
M
m
(1)
where C denotes an aqueous concentration, M is the amount
adsorbed, K
L
is the Langmuir constant, which represents the
afnity of the adsorption process, and M
m
is the maximum
amount adsorbed as C increases. Aplot of C/Mversus C should
give a straight line of slope 1/M
m
and an intercept of 1/K
L
M
m
on the C/M axis.
For comparison, we also t the EGCG adsorption isotherm
with the Freundlich model, an empirical exponential equa-
tion, which can be described as
12
M K
f
C
1=n
(2)
where M is the amount adsorbed, 1/n is a constant, K
f
is an
afnity constant, and C is the concentration of the EGCG
solution. Its logarithmic form is
log M log K
f
log C=n (3)
i.e. a plot of log M versus log C should give a straight line of
slope 1/n and an intercept of log K
f
on the log M axis.
2.6. Fluorescence quenching measurements
Fluorescence quenching is described by the SternVolmer
equation
13
:
F
0
F
1 k
q
t
0
1 K
SV
Q (4)
In this equation F
0
and F are the uorescence intensities in
the absence and presence of a quencher, respectively, k
q
is the
bimolecular quenching constant, t
0
is the lifetime of the
uorophore in the absence of quencher, and [Q] is the
concentration of the quencher. If the quenching is known
to be dynamic or static, the SternVolmer constant (K
SV
) will be
represented by dynamic quenching constant (K
D
) or static
quenching constant (K
S
). Otherwise this constant will be
described as K
SV
. Quenching can also occur as a result of the
formationof a non-uorescent ground-state complexbetween
the uorophore and the quencher, and in such cases, k
q
is
calculated. For biomacromolecules, the lifetime of the
uorophore is approximately 10
8
s, and the maximum value
possible for diffusion-limited quenching in water is
10
10
M
1
s
1
.
13
Quenching data are usually presented as
plots of F
0
/F versus [Q]. This is because F
0
/F is expected to be
linearly dependent upon the concentration of the quencher. A
plot of F
0
/F versus [Q] yields an intercept of one on the y-axis
and a slope equal to K
SV
. A linear SternVolmer plot is
generally indicative of a single class of uorophore in proteins,
all equally accessible to the quencher. This means that only
one mechanism (dynamic or static) of quenching occurs.
When the value of the bimolecular quenching constant is
much higher than that of the diffusion-limited quenching
constant, and the SternVolmer plot presents as linear, it
could mean that there is complex formation between protein
and quencher, corresponding to a static mechanism.
The uorescence intensities were recorded with a
PerkinElmer LS5 luminescence phosphorescence spectro-
photometer, using 2.5 nm excitation; 5 nm emission slit
widths; the lamp voltage, 700 V; scan velocity, 1200 nm s
1
.
A 1-cm quartz cell was used. To determine the linear
concentration range for WS and PS uorescence, a series of
saliva solutions with increasing concentrations (01 mg/ml)
were prepared in ion exchanged water at pH = 7.0 and
25 0.01 8C. The maximum excitation wavelength (l
ex
) and
maximum emission wavelength (l
em
) for saliva were 285 nm
and 348 nm, respectively. The linear range of saliva
uorescence was between 0.01 mg/ml and 0.10 mg/ml (data
were not shown). Therefore, a 0.5 mg/ml salivary protein
was chosen as the concentration for uorescence quenching
in this experiment.
Saliva (90 ml) was mixed with different amounts (10 ml,
20 ml, 30 ml, 40 ml, 50 ml, and 60 ml) of the 3 mM EGCG solution,
in a total of 3 ml, respectively. The change in uorescence
emission intensity was measured within 1 min of adding the
EGCGsolutionto saliva at 25 0.01 8C. Eachmeasurement was
repeated in triplicate and the mean and standard deviation
were calculated. For the calculation of the quenching
constants, the data were plotted as a SternVolmer plot of
a r c hi v e s o f o r a l b i o l og y 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 472
F/F
0
against [Q], and the quenching constant was calculated by
linear regression.
Atea polyphenol possesses intrinsic uorescence at the l
ex
(282 nm) and displays a corresponding l
em
at 318 nm
13
which
is near those of salivary proteins. To eliminate the background
effect on the uorescence quenching values of salivary
proteins, the uorescence emission intensities of EGCG were
measured at 318 nm as a blank titration series (i.e. adding
0.25 ml of each EGCG concentration into 3 ml of buffer). The
uorescence values obtained were then subtracted from the
uorescence intensity values obtained for saliva quenching.
2.7. Statistical analysis
The data were analyzed for statistical signicance using a two-
way ANOVA analysis followed by a SNK-q test for multiple
comparisons or a paired t-test for two sample comparisons
(a = 0.05).
3. Results
3.1. FTIR spectra for EGCG on WS and PS surfaces
The FTIR spectrum can be used to identify adsorption
processes onto the surface, and to prove the existence of
hydrogen bonding between the adsorbed EGCG molecules and
saliva surfaces. The FTIR spectra of WS and PS surfaces with
and without the EGCGadsorptionwere obtained(Figs. 2 and 3).
The FTIR spectra of WS and PS surfaces display two bands at
1660 cm
1
, 1623 cm
1
, and 1550 cm
1
; this is characteristic of
protein amide bands.
11
The 1660 cm
1
and 1623 cm
1
(amide I)
bands were attributed to protein amide C O stretching
vibrations and NH
3
+
bending vibrations, and the 1550 cm
1
(amide II) band was due to the amide NH bending vibrations
and CN stretching vibrations.
11
The peak of the infrared
absorption of amide bands I and II for the EGCG adsorbed on
WS and PS surface had no other remarkable shifts at
1660 cm
1
, 1623 cm
1
, and 1550 cm
1
. New peaks at
1727 cm
1
, 1525 cm
1
, 1319 cm
1
, and 1038 cm
1
in the FTIR
spectra of EGCG adsorbed on WS surfaces and at 1727 cm
1
,
1391 cm
1
, 1230 cm
1
, and 1014 cm
1
in the FTIR spectra of
EGCG adsorbed on PS surfaces were observed, which illumi-
nate the characteristic bands of EGCG.
3.2. Mass, thickness and viscoelasticity of EGCG adlayer
Figs. 4 and 5 show the recorded the time-resolved frequency
change (DF) and energy dissipation change (DD) at different
concentrations of EGCG solution (from2 mMto 24 mM) for the
fth overtone during EGCG adsorption to WS (Fig. 4) and PS
(Fig. 5) surfaces for 70 min. There was an obvious decrease in
Fig. 2 Infrared spectra of the WS-modified quartz crystal
surface before (black line) and after (gray line) EGCG
adsorption.
Fig. 3 Infrared spectra of the PS-modified quartz crystal
surface before (black line) and after (gray line) EGCG
adsorption.
Fig. 4 Shifts of time-dependent frequency and energy
dissipation for EGCG adsorption on the WS-modified
quartz crystal surface at various EGCG concentrations: (a)
2 mM; (b) 4 mM; (c) 8 mM; (d) 12 mM; (e) 16 mM; and (f)
24 mM, at pH = 7.0 and 25 W0.01 8C.
a r c hi v e s o f or a l b i ol o gy 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 473
DF and marked increase in DD right after the injection of EGCG
solution. This was followed by a more gradual change in DF
and DD until the steady state was reached. In order to describe
EGCG adsorption behaviours accurately, the values of mass,
thickness, elasticity, and viscosity of the EGCG adlayer on the
saliva surfaces at various concentrations of EGCG solution
were calculated by the Sauerbrey equation and by the Voigt
model (Fig. 6). The mass, thickness, elasticity, and viscosity
of the EGCG adlayer on WS surfaces increased with the
increase in EGCG concentration. The mass and thickness of
the EGCG adlayer on PS surfaces increased with the increase
in EGCG concentration, but the elasticity and viscosity of the
EGCG adlayer on PS surfaces decreased along with the
increase in EGCG concentration. At various concentrations
of EGCG solution, the values of mass and thickness for the
EGCG adlayer on WS surfaces were signicantly higher than
those for the EGCG adlayer on PS surfaces (P < 0.05). The
values of elasticity and viscosity of the EGCG adlayer on WS
surfaces were distinctly lower thanthose of the EGCGadlayer
onto PS surfaces at 2 mM and 4 mM concentrations of EGCG
solution (P < 0.05), and signicantly higher at 16 mM and
24 mM concentrations (P < 0.05), but similar at 8 mM and
12 mM concentrations (P > 0.05).
3.3. Adsorption models and constants
The data of EGCGadsorption on WS and PS surfaces were t to
the Langmuir model and to the Freundlich model with the
Fig. 5 Shifts of time-dependent frequency and energy
dissipation for EGCG adsorption on the PS-modified quartz
crystal surface at various EGCG concentrations: (a) 2 mM;
(b) 4 mM; (c) 8 mM; (d) 12 mM; (e) 16 mM; and (f) 24 mM, at
pH = 7.0 and 25 W0.01 8C.
Fig. 6 Changes of mass (A), thickness (B), elasticity (C), and viscosity (D) of the EGCG adlayer on WS and PS surfaces at
various EGCG concentrations. *P < 0.05 versus the other groups (ANOVA).
a r c hi v e s o f o r a l b i o l og y 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 474
correlationcoefcient of determination, as shownin Fig. 7 and
Table 1. Though both models can be used to describe the
adsorption behaviour, the values of R
2
for EGCG adlayer onto
WS and PS surfaces from the Freundlich model were all
signicantly higher than those from the Langmuir model
(P < 0.05), which suggests that using the Freundlich model to
t the experimental data is much more satisfactory compared
with the Langmuir model. The calculated adsorption con-
stants from the Langmuir and the Freundlich model are listed
in Table 2. All of the values of the EGCG adsorption constants
between WS and PS surfaces display signicant differences
(P < 0.05), which indicate that the afnity of EGCG to WS is
obviously higher than that of EGCG to PS.
3.4. Temperature-dependent measurement by QCM-D
EGCG adsorption on WS and PS surfaces were performed by
temperature-dependent QCM-D measurements for 12 mM
EGCG at 25 8C, 30 8C, and 35 8C, as shown in Fig. 8. By
increasing the temperature, the mass of the EGCG adsorption
on WS or PS surfaces increased, namely, a higher mass of the
EGCG adlayer was observed at higher temperatures (P < 0.05).
3.5. Experiment of uorescence quenching
The binding afnities for EGCG to WS and to PS were also
evaluated by the measurement of the intrinsic uorescence
intensity of salivary proteins before and after the addition of
different concentrations of EGCG (Fig. 9). In all cases, a
decrease in the uorescence intensity caused by quenching
was observed, however, there was no shift of the maximum
l
em
. EGCG was found to eventually lead to about 55%
quenching of WS and PS uorescence emission intensity.
Fig. 10 shows the SternVolmer plots of EGCG quenching WS
and PS intrinsic uorescence. K
SV
was determined by a linear
regression of a plot of F
0
/F against [Q]. Hence, k
q
can be
Fig. 7 Adsorption isotherm of EGCG onto WS (shown in
* and solid line) and PS (shown in * and dashed line)
surfaces fitted to the Langmuir model (A) and the
Freundlich model (B).
Table 1 The correlation coefficient of determination (R
2
)
for the Langmuir model and the Freundlich model.
Saliva Langmuir model Freundlich model
WS 0.966 0.001
a
0.994 0.003
c
PS 0.902 0.013
b
0.958 0.015
d
Within the same column, the values (MV SD) with different
superscript letters are signicantly different (ANOVA, P < 0.05).
Table 2 The adsorption isotherm constants of the
Langmuir model and the Freundlich model.
Saliva M
m
(ng/cm
2
) K
L
(mM
1
) K
f
(ng/cm
2
mM)
WS 484 14
a
260 18
a
151 8
a
PS 366 16
b
131 31
b
79 19
b
Within the same column, the values (MV SD) with different
superscript letters are signicantly different (paired t-test,
P < 0.05).
Fig. 8 Mass changes for 12 mM EGCG adsorption on WS
and PS surfaces at various temperatures. *P < 0.05 versus
the other groups (ANOVA).
a r c hi v e s o f or a l b i ol o gy 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 475
calculated by the ratio between K
SV
and t
0
from Eq. (4). Table 3
summarizes the calculatedK
SV
andk
q
. The values of K
SV
and k
q
for EGCG binding to WS are signicantly higher than those to
PS (P < 0.05).
4. Discussion
4.1. Property of EGCG adlayer
The better t of the EGCG adsorption isotherm using the
Freundlich model rather than the Langmuir model conrmed
that the distribution of EGCG binding on saliva surfaces was
due to the heterogeneity and continuous binding sites, which
are two characteristics of the Freundlich model.
11
The
heterogeneous distribution of the EGCG adlayer is due to
the aggregation of saliva through EGCG bridges because some
characteristic bands of FTIR spectra for EGCG were observed.
The increases of the mass, thickness, and viscoelasticity of the
EGCG adlayer on WS surfaces were observed when the
concentration of the EGCG solution was increased from
2 mM to 24 mM, which suggests that the EGCG adlayer
eventually became more rigid. At low EGCG concentrations,
a small fraction of water may be entrapped internally in the
EGCG adlayer, and result in a more soft and incompact EGCG
adlayer on WS surfaces. At high EGCG concentrations, some
adsorbed EGCG molecules may act as bridges to allow salivary
molecules to aggregate with each other. At the same time,
water, coupled with salivary molecules, would be gradually
driven out during the EGCG adsorption. Consequently, the
greater mass and thickness of EGCG adsorbed on WS surface
will cause anincrease inboththe elasticity and viscosity of the
EGCG adlayer. Therefore, the EGCG adlayer will become more
compact and stiff. The same phenomena were taking place in
the adsorption of the EGCG adlayer on BSA surface.
11,14
Even if
the mass and thickness of the adsorbed EGCG adlayer on PS
surfaces at low EGCG concentrations were lower compared
with those at high EGCG concentrations, the elasticity and
viscosity of the EGCG adlayer at low concentrations were
higher than those at high concentrations. The tendency of the
viscoelasticity of the EGCG adlayer on PS surfaces was just the
opposite to that on WS surfaces, whichsuggests that the EGCG
adlayer on PS surfaces at low EGCG concentrations was
adsorbed more completely, and was relatively more rigid due
to the strong interactions between EGCG and PS. Considering
that the total concentrations of salivary proline-rich proteins
(PRPs) and histatins are nearly three times as much compared
to that of a-amylase (0.66 mg/ml) in PS,
4
it is reasonable to
think that PRPs and histatins may be the main proteins
participating in the strong interaction between EGCG and PS,
forming a stiffer and more compact adlayer at low EGCG
concentrations. The increasing trend of mass and thickness
for the EGCG adlayer on WS surfaces is similar to that on PS
surfaces, but the amount of adlayer on WS surfaces is
signicantly higher than those on PS surfaces at various
Fig. 9 Fluorescence emission spectra of WS and PS in the
absence and the presence of different concentrations of
EGCG: (a) 0 mM; (b) 2 mM; (c) 4 mM; (d) 8 mM; (e) 12 mM;
(f) 16 mM; and (g) 24 mM.
Fig. 10 SternVolmer plots of fluorescence quenching of
WS (shown in * and solid line) and PS (shown in *
and dashed line) at different concentrations of EGCG.
Table 3 SternVolmer (K
SV
) and bimolecular quenching
(k
q
) constants for interaction of EGCG with WS and PS.
Saliva k
q
(10
12
M
1
s
1
) K
SV
(10
4
M
1
)
WS 2.006 0.022
a
2.006 0.222
a
PS 1.907 0.008
b
1.907 0.008
b
Within the same column, the values (MV SD) with different
superscript letters are signicantly different (paired t-test,
P < 0.05).
a r c hi v e s o f o r a l b i o l og y 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 476
concentrations of EGCGsolution. The mainreasonis related to
the gel-like network structure from the mucins in WS. In
general, the association of EGCG with saliva will reduce the
salivary lubrication. The astringency is perceived as the
friction between two non-lubricated surfaces. The increased
friction induces a tactile sensation by activating mechanor-
eceptors in the mouth, thus leading to the perception of
astringency.
1517
Withmore EGCGdeposited onsaliva surfaces
by the continuous binding sites and aggregation capacity of
saliva through EGCG bridges, the viscoelasticity and coverage
of the EGCG adlayer on the oral mucosal surface became
larger. Thus the astringency and loss of lubrication of the oral
mucosa would be likely to aggravate the mucosa. Therefore,
we can make a reasonable conclusion that the viscoelasticity
of the EGCG adlayer ties up with an unpleasant sensation of
roughness and dryness, i.e. astringency in the oral cavity. This
discomfort, however, is a subjective sensation that is
complicated to quantify, and measurement of its gradual
elimination by an instrument remains an elusive target in the
future.
4.2. Afnity of EGCG to saliva
In our adsorption studies by QCM-D, the values of M
m
, K
L
and
K
f
for EGCG on WS surfaces are all signicantly higher than
those on PS surfaces, which suggests that the binding afnity
of EGCG to WS is higher than that to PS. A possible reason for
this discrepancy is that the oral mucosa is a broad binding site
and a less permeable barrier for all experimentally proven
polyphenols and is, therefore, an efcient tissue for the
absorption of topically consumed dietary polyphenols.
18
The
saliva lm-coated tooth, mucosa surface, and soluble saliva
are probably sites of biological action. Because WS contains
many more complex constituents, more binding sites will be
exposed to WS surfaces bringing on the signicantly different
binding afnities between EGCG to WS and to PS. This result
suggests that WS has a much stronger neutralization for the
harmful effects of EGCG than PS. The high afnity of EGCG to
saliva suggests that the precipitation of EGCG/saliva is not
easy to dissociate. This result also implies that the EGCG
adlayer has a good retention associated with saliva on the
mucosa.
In order to gain further support for the binding afnity of
soluble saliva with EGCG, quenching of the tryptophan
uorescence of salivary proteins by titration with an EGCE
solutionwas carriedout. Inproteins, the three aromatic amino
acids, i.e. phenylalanine (Phe), tyrosine (Tyr), and tryptophan
(Typ), are all uorescent. WS contains a complex mixture of
some 330 proteins and peptides, whichinclude numerous Phe,
Tyr, and Trp residues. For instance, a-amylase, PRPs and
histatins contain 18 Trp, 20 Tyr and 28 Phe; 622 Tyr, no Trp
and Phe; and2 Tyr, no Trp andPhe amino acid residues intheir
sequences, respectively (NCBI Protein Data Bank). To explain
the data from uorescence quenching in this experiment, it is
important to understand what kind of interaction occurs
between EGCG and salivary proteins by calculating the
bimolecular quenching constant. In these studies, the
SternVolmer plots for EGCGquenching of WS and PS proteins
are all linear (Fig. 10), and the values of k
q
for WS and PS are all
100-fold higher than the maximum value of diffusion-limited
quenching in water (10
10
M
1
s
1
). This means that only a
static quenching occurred,
19
and a ground-state complex
formed between EGCG and salivary protein. The values of K
SV
are equal to those of K
S
(Table 3) corresponding to a static
mechanism. The value of K
S
for the EGCG quenching of WS is
larger than PS, which reects that the afnity of EGCGto WS is
higher than that to PS. The results of the binding afnity of the
uorescence quenching are consistent with those of EGCG
adsorption by QCM-D, suggesting that both experiments, i.e.
uorescent quenching and QCM-D, are useful methods to
study the afnity between polyphenols and proteins. Consid-
ering the biological functions of soluble salivary proteins, the
binding of soluble saliva to EGCG will directly counteract with
saliva, providing local protection at the oor of the mouth
against mechanical forces during eating and speaking.
When considering the effect of EGCG quenching on the
uorescence spectra of WS and PS, no apparent l
em
shift was
observed, which suggested no other changes in the local
environment of the tryptophan residues. Except for the
evidence that EGCG was adjacent to the tryptophan residues
at the moment of excitation, the molecular conformation of
the protein was not affected no matter what the EGCG
mechanism of interaction was. If there had been a conforma-
tional change for WS and PS proteins, a shift in the peak of
amide band I or disappearance of the peak corresponding to
the NH residual amide band II would have been observed,
however, neither of themoccurred. The biological functions of
saliva have been shown to be dependent upon the intact
conformation of proteins. The high conformational stability of
the native state after salivary proteins were quenchedby EGCG
suggested that the molecular structure of salivary proteins
would be maintained, along with the native biological role and
activity. One experimental data showed that conformational
changes of WS proteins were observed by the exposure of their
tryptophan residues to catechin and epicatechin.
1
The
reasonable explanation is that EGCG has a higher molecular
weight compared with epicatechin and catechin, but phenolic
compounds with low molecular weights had been reported to
affect boththe secondary and tertiary structure of the proteins
as determined by circular dichroism and FTIR.
20
4.3. Driving force of EGCG binding to saliva
Although EGCG bridges may cause aggregation of salivary
proteins,
21,22
hydrogen bonding between the phenolic hydrox-
yl groups of EGCG and the amide groups of saliva as well as
hydrophobic interactions may also occur, and are responsible
for EGCG binding to saliva. Hydrogen bonding was conrmed
by our measurement of the FTIR spectra. The hydrophobic
interaction was veried by temperature-dependent QCM-D
measurements. At increased temperature, hydrophobic inter-
actions are known to be favored.
11
In our study, EGCG has the
characteristic of temperature-dependent precipitation, as a
higher mass of the EGCG adlayer was observed at higher
temperatures. Increasing the temperature may cause a partial
unfolding of molecules of salivary proteins, thus increasing
the exposure of the hydrophobic surfaces of the proteins to
which more EGCG molecules may take part in binding
reactions.
11,18
The entropically driven characteristic of EGCG
binding on saliva suggests that EGCG may preferentially bind
a r c hi v e s o f or a l b i ol o gy 5 5 ( 2 0 1 0 ) 4 7 0 4 7 8 477
to hydrophobic constituents of the oral mucosa rather than
soluble salivary proteins, and that binding to these compo-
nents may initiate the astringent response. Therefore, the
hydrogen bonding, the hydrophobic interactions, and the
entropy-drive may be the main driving force of EGCG binding
to saliva.
5. Conclusion
To our knowledge, this study is the rst investigation on the
dynamic process of EGCG binding on saliva by QCM-D and
uorescence quenching. The results suggest that the astrin-
gency and loss of lubrication in the oral cavity may be
attributed to the viscoelasticity of EGCG adlayer on the oral
mucosa, the afnity of EGCG to saliva, and the entropy-driven
characteristic of EGCG. Hence, the null hypothesis is rejected.
The hydrogen bonding, the hydrophobic interactions, and the
entropy-drive are the main driving forces for EGCG binding to
saliva, as indicated by the FTIR spectra and the temperature-
promoted EGCG adsorption. The afnities of EGCG to saliva
suggest that whole saliva has a much stronger neutralization
to the effect of EGCG than parotid saliva.
Acknowledgements
We are grateful to Prof. Qi-Fu Li (Department of Biochemistry
and Biotechnology, School of Life Sciences, Xiamen University,
China) for running the adsorption properties of saliva analysis.
The authors also thank the National Natural Science Founda-
tion of China (Grants No.: 20773100) for the nancial supports.-
Funding: None.Competing interests: We declare that we have no
nancial and personal relationships with other people or
organizations that can inappropriately inuence our work,
there is no professional or other personal interest of any nature
or kind in any product, service and/or company that could be
construed as inuencing the position presented in, or the
reviewof, themanuscript entitled.Ethical approval: Not required.
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