Full text of DOI:10.1111/j.1348-0421.2002.tb02693.x

Microbiol. Immunol., 46(4), 249―255, 2002
Inhibition by Apple Polyphenols of
ADP-Ribosyltransferase Activity of Cholera Toxin
and Toxin-Induced Fluid Accumulation in Mice
Takao Saito1, 2, Masami Miyake1, Masamichi Toba2, Hiroshi Okamatsu2, Seiichi Shimizu3,
and Masatoshi Noda*, 1
1Department of Molecular Infectiology, Graduate School of Medicine, Chiba University, Chiba, Chiba 260
Nutraceuticals Research Institute, Otsuka Pharmaceuticals Co., Ltd., Otsu, Shiga 520
―
8670, Japan, 2Otsu
3
―
0002, Japan, and P-14 Project,
Otsuka Pharmaceuticals Co., Ltd., Osaka, Osaka 540―0021, Japan
Received October 12, 2001. Accepted January 5, 2002
Abstract: The effects of crude polyphenol extracted from immature apples on the enzymatic and biological
activities of a cholera toxin (CT) were investigated. When the apple polyphenol extract (APE) was exam-
ined for properties to inhibit CT-catalyzed ADP-ribosylation of agmatine, it was found that APE inhibit-
ed it in a dose-dependent manner. The concentration of APE to inhibit 50% of the enzymatic activity of CT
(15 ꢀg/ml) was approximately 8.7 ꢀg/ml. The APE also diminished CT-induced fluid accumulation in two
diarrhea models for in vivo mice. In the ligated ileum loops, 25 ꢀg of APE significantly inhibited fluid accu-
mulation induced by 500 ng of CT. In a sealed mouse model, even when APE was administered orally 10
min after a toxin injection, fluid accumulation was significantly inhibited at a comparable dosage.
Lineweaver-Burk analysis demonstrated that APE had negative allosteric effects on CT-catalyzed NAD:
agmatine ADP-ribosyltransferase. We fractionated the APE into four fractions using LH-20 Sephadex resin.
One of the fractions, FAP (fraction from apple polyphenol) 1, which contains non-catechin polyphenols, did
not significantly inhibit the CT-catalyzed ADP-ribosylation of agmatine. FAP2, which contains com-
pounds with monomeric, dimeric, and trimeric catechins, inhibited the ADP-ribosylation only partially, but
significantly. FAP3 and FAP4, which consist of highly polymerized catechin compounds, strongly inhibit-
ed the ADP-ribosylation, indicating that the polymerized structure of catechin is responsible for the
inhibitory effect that resides in APE. The results suggest that polymerized catechin compounds in APE inhib-
it the biological and enzymatic activities of CT and can be used in a precautionary and therapeutic man-
ner in the treatment of cholera patients.
Key words: Apple polyphenols, Cholera toxin, ADP-ribosyltransferase, Fluid accumulation
Cholera is a disease caused by the colonization of
Vibrio cholerae in the upper intestinal tract, which caus-
es massive secretory diarrhea, severe dehydration, and
circulatory collapse (16). Severe dehydration is respon-
Health and Welfare every year from 1990 through 1999
(5).
A major virulence factor of V. cholerae is the cholera
toxin (CT), a proteinaceous toxin produced by the bac-
terium (1). The toxin consists of one enzymatic A sub-
unit and five B subunits. The A subunit has ADP-ribo-
syltransferase (ART) activity, which mono-ADP-ribo-
sylates a mammalian GTP-binding regulatory protein,
Gsα, of the adenylate cyclase system. The A subunit
associates with the homo-pentameric B-oligomer, which
sible for 20
―
45% of the deaths of patients who do not
receive rehydration therapy (2). According to a report of
the World Health Organization (21), 254,310 cases and
9,175 deaths due to cholera were reported worldwide in
1999. Cholera continues to be a major public health
problem in developing countries. In Japan, cases of
imported infection have been reported by the Ministry of
Abbreviations: ADP, adenosine 5'-diphosphate; APE, apple
polyphenol extract; ART, ADP-ribosyltransferase; CT, cholera
toxin; FA, fluid accumulation; FAP, fraction from apple polyphe-
nol; GTP, guanosine 5'-triphosphate; Km, Michaelis constant;
NAD, nicotinamide adenine dinucleotide; SD, standard deviation;
TLC, thin-layer chromatography.
*Address correspondence to Dr. Masatoshi Noda, Department
of Molecular Infectiology, Graduate School of Medicine, Chiba
University, 1
―8
―
1 Inohana, Chuo-ku, Chiba, Chiba 260
―8670,
Japan. Fax: 81
u.ac.jp
―
43 226 2049. E-mail: noda@med.m.chiba-
―
―
249

250
T. SAITO ET AL
mediates the binding of the toxin to a specific receptor,
GM1 monosialoganglioside, on the surface of target
eukaryotic cells (3). The bound toxin is then endocytosed
in endosomal vesicles and transported intracellularly
through the Golgi apparatus to the endoplasmic reticu-
lum, where the A subunit is translocated out of the vesi-
cle into cytosol (11). It is believed that the translocated
A subunit moves to the inner surface of the plasma
membrane and ADP-ribosylates Gsα, resulting in con-
stitutive activation of the adenylate cyclase. The intox-
ication of intestinal epithelial cells by the toxin causes the
accumulation of intracellular cAMP, which stimulates
chloride secretion from and inhibits neutral sodium chlo-
ride absorption into intestinal mucosa, consequently
leading to the severe secretory diarrhea observed in
cholera patients (1, 3).
solved in distilled water and applied to a Sephadex LH-
20 (Amersham Pharmacia Biotech K.K.) column. By
elution with methanol, three distinct fractions, FAP1,
FAP2, and FAP3, were recovered from the column in this
order. An additional elution with acetone-water (1:1)
resulted in the recovery of FAP4. The polyphenol con-
tents of these fractions were identified by thin-layer
chromatography (TLC) using Kieselgel 60 as a TLC
plate and Benzene-HCOOEt-HCOOH (1:7:1) as a devel-
oping system (4). The indications were that FAP1 main-
ly contained non-catechin polyphenols and that FAP2,
FAP3, and FAP4 contained monomeric to trimeric cate-
chins, oligomeric catechins, and polymeric catechins,
respectively. From 300 mg of apple polyphenol, 105 mg,
77 mg, 37 mg, and 57 mg of evaporated FAP1, FAP2,
FAP3, and FAP4 were obtained, respectively.
Since CT is a major factor responsible for the induc-
tion of diarrhea in cholera, it is expected that inactivation
of CT-derived ADP-ribosyltransferase activity in the
intestinal tract would block the onset of the disease,
leading to a decrease in the mortality rate of cholera
patients (17). Toda et al. (19) have reported that tea
polyphenol inhibited the fluid accumulation induced by
CT in sealed adult mice. Effects of other polyphenols,
however, were not elucidated until recently. In the pres-
ent study, we describe how a crude polyphenol fraction
extracted from immature apples inhibits both the enzy-
matic and biological activities of CT. This suggests the
possibility that oral administration of an apple polyphe-
nol can be used in cholera patients as a precautional
and therapeutic procedure.
ADP-ribosyltransferase activity. To quantify the
ADP-ribosyltransferase activity of CT, an agmatine
assay was performed as described (13). The reaction was
started by the addition of a reaction buffer containing
[14C]NAD to 3 µg of CT and several amounts of APE,
FAPs, or other polyphenols, in a total volume of 200 µl.
After incubation for 3 hr at 30 C, a 100 µl portion of the
reaction mixture was applied to an AG1-X2 column
(5×45 mm). The unbound materials were eluted with
distilled water and collected in scintillation vials. The
radioactivity in the eluate was measured with a liquid
scintillation counter.
Fluid accumulation in the ileal loop assay. A mouse
ileal loop assay was performed as described previously
(6). Four-week-old male ICR mice (JAPAN SLC., Inc.)
weighing 20 to 25 g were used. The mice were starved
overnight with free access to water and anesthetized
with sodium pentobarbital, and their intestines were
exteriorized through a midline incision. One ligated
intestinal segment (loop) about 2 cm long was created per
intestine. Five hundred ng of CT and/or APE (total
volume 100 µl) was simultaneously injected into the
loop. In 6 hr, the mice were killed, and the loop was
removed from the carcass. The degree of fluid accumu-
lation was expressed as the ratio of loop content (weight)
to loop length.
Materials and Methods
Chemicals. Crude apple polyphenol extract (Apple-
phenon SH) was purchased from Nikka Whisky Dis-
tilling Co., Ltd. (＋)-Catechin, phloretin, phloridzin, p-
coumaric acid, and chlorogenic acid were from Sigma-
Aldrich Japan K.K. Procyanidin B1, procyanidin B2,
procyanidin C1, and (－)-epicatechin were from the
Funakoshi Co., Ltd.
CT was purchased from the List Biological Labora-
tories; [adenine-U-14C]NAD (250
―
300 mCi/mmol), from
Fluid accumulation in the sealed mice model. The
mice were starved for 20 hr with free access to water
before the experiments. Each mouse was intragastrically
given 0.2 ml of a 3% sodium-hydrogen-carbonate solu-
tion containing 20 µg of CT. In 10 min, 0.1 ml of test
agents was intragastrically administered to the mice.
After a 6-hr incubation, the mice were sacrificed. The
whole intestine (from the pyloric valve to the rectum) was
resected and weighed. The fluid accumulation (FA)
value was calculated by the following formula:
Amersham Pharmacia Biotech K.K.; AG1-X2 resin (200
―
400 mesh), from Nippon Bio-Rad Laboratories; and
pentobarbital, from the Dainippon Pharmaceutical Co.,
Ltd. All other reagents were either from Wako Pure
Chemical Industries, Ltd. or from Sigma-Aldrich Japan
K.K.
Preparation of FAPs. Fractionation of APE into four
FAPs (fractions from apple polyphenol) was kindly car-
ried out by Dr. Junei Kinjo (Fukuoka University) by
the established procedure (18). Briefly, APE was dis-
FA value＝A÷(B－A)×1,000, where A represents

251
CHOLERA TOXIN INHIBITOR
Fig. 1. Inhibitory effects of APE on CT-catalyzed ADP-ribosy-
lation. Three µg of CT (15 µg/ml) was incubated for 3 hr at 30 C
with agmatine in the presence of the indicated concentrations of
APE. Values represented are the mean and standard deviations
from 3 determinations.
Fig. 2. Inhibitory effects of APE on the CT-induced fluid accu-
mulation in the mouse ileal loop assay. Each loop was injected
with 0.1 ml of a solution containing 500 ng of CT and/or 12.5 µg,
25 µg, or 50 µg of APE. In 6 hr, mice were sacrificed, and the
loop was removed from the carcass. The degree of fluid accu-
mulation was expressed as the ratio of loop content (weight) to
loop length. Values represented are the mean and standard devi-
ations from five animals. These values were compared with CT
alone by the Dunnett’s two-tailed test (*: P＜0.01).
the weight of the excised gut and B represents the body
weight of the mouse.
Statistical analysis. Data were expressed as means±
SD, and the differences in the groups were analyzed
using one-way ANOVA with Dunnett’s multiple com-
pared test. A P value of ＜0.05 was considered statisti-
cally significant. All statistical analyses were performed
using SAS ver.6.12 (SAS Institute).
Results
(Fig. 2). The result indicates that APE not only inhibits
the enzymatic activity of the CT but also disturbs its
biological activity in vivo.
Inhibitory Effect of APE on CT-Catalyzed ADP-Ribosy-
lation of Agmatine
The inhibitory effect of APE on CT-induced fluid
accumulation was also confirmed in the sealed mouse
model, in which toxin and APE were administered per
os. In this model, CT was intragastrically delivered
through the mouth and esophagus using thin tubing. At
10 min after toxin administration, APE was delivered by
the same route. When 10 mg or 5 mg of APE was
given to the mice, the FA values decreased by 60% of the
value from the positive control (CT alone, Fig. 3). The
degree of the decrease was statistically significant. The
results indicate that, even when APE ingestion was made
10 min after CT administration, it still inhibited CT-
induced fluid accumulation.
The enzymatic A subunit of CT catalyzes the transfer
of ADP-ribose from NAD to agmatine and other simple
guanidino compounds (13). We examined the effect of
APE on the CT-catalyzed ADP-ribosylation of agmatine.
When APE was added in the assay mixture, it inhibited
the ADP-ribosylation in a dose-dependent manner (Fig.
1). This result indicates that APE inhibits the enzy-
matic activity of CT. Under this condition, the concen-
tration of APE to inhibit 50% of CT-catalyzed ADP-ribo-
sylation was estimated to be 8.7 µg/ml.
Effect of APE on CT-Induced Fluid Accumulation in
Mice
Since one of the biological activities of CT is the
induction of fluid accumulation in the ileal tract, the
inhibitory effects of APE on the CT-induced fluid accu-
mulation in a mouse intestinal-loop assay were investi-
gated. When a 500 ng/loop of CT was inoculated,
approximately 17 mg of fluid accumulation/cm in the
loop was observed, indicating the diarrhegenic activity of
CT in this animal model. When APE at concentrations
higher than 25 µg/ml was simultaneously injected into
the loop, the fluid accumulation was significantly reduced
Qualification of Inhibitory Components in APE
Kanda and his collaborators reported the polyphenol
composition in APE, which they had analyzed by high
performance liquid chromatography (9). According to
their report, APE consists of chlorogenic acid (20%,
dry weight/dry weight), phloridzin (4%), phloretin (1%),
p-coumaric acid (1%), caffeic acid (3%), (－)-epicatechin
(6%), (＋)-catechin (0.5%), procyanidin B1 (3%), pro-
cyanidin B2 (7%), oligomeric and polymerized cate-
chins (40%), and unknown residual compounds (15%).

252
T. SAITO ET AL
Table 1. Qualification of inhibitory fractions in APE
Sample
Inhibition (%)
Components
Control
APE
0.0±4.4
95.4±0.7
—
—
FAP1
FAP2
FAP3
FAP4
3.5±5.2
39.4±5.8
98.3±1.5
95.5±0.3
Non-catechin polyphenols
Mono- to trimeric catechins
Oligomeric catechins
Polymeric catechins
CT at 15 µg/ml was incubated for 3 hr at 30 C with agmatine
14
and [ C]NAD in the presence or absence of a concentration (25
µg/ml) of APE and FAPs. The values represented are the mean
and standard deviations from three determinations.
significantly, but procyanidin B1, procyanidin B2 (dimer-
ic catechins), and procyanidin C1 (trimeric catechin)
showed relatively higher inhibitory activity at the same
concentration (2.4±3.3%, 0.1±5.3%, 17.4±12.7%,
9.8±10.5%, and 25.4±5.5%, respectively). Based on
these results, together with the polyphenol composition
in APE, it is concluded that highly polymerized catechins
in FAP3 and FAP4 are the major inhibitory polyphenols
in APE and that the non-catechin type and monomeric
catechins exhibit a very slight effect, if any, on CT-cat-
alyzed ADP-ribosylation.
Fig. 3. Inhibitory effects of APE on CT-induced fluid accumula-
tion (FA) in sealed mice model. After administration of CT for 10
min, the mice were given 5 mg or 10 mg of APE. After a 6-hr
incubation, the mice were sacrificed. The whole intestine was
resected and weighed. The FA value was determined as a ratio of
the body weight minus the intestine weight to the intestine
weight. Values represented are the mean and standard devia-
tions, *: P＜0.05).
In order to identify which polyphenols in APE are main-
ly responsible for the inhibitory effect on CT-catalyzed
ADP-ribosylation, we fractionated the APE-derived
polyphenols into four different fractions and compared
their ability to inhibit the ADP-ribosyltransferase activ-
ity of CT. One of the fractions, FAP1, which contains
non-catechin-type polyphenols such as chlorogenic acid,
phloridzin, phloretin, p-coumaric acid, and caffeic acid,
did not inhibit the ADP-ribosylation of agmatine at 25
µg/ml (Table 1). The inhibitory activity of FAP2, which
contains mono- to trimeric catechins [(－)-epicatechin,
(＋)-catechin, procyanidin B1, procyanidin B2, and pro-
cyanidin C1], was significantly higher than that of FAP1,
but only 40% of the transferase activity was inhibited by
the fraction. FAP3 and FAP4, which contain highly
polymerized (tetrameric or higher) catechin compounds,
strongly inhibited the enzymatic activity of CT, sug-
gesting that highly polymerized catechins are the major
inhibitory components in APE (Table 1). In the next
step, we obtained the isolated polyphenols from com-
mercial sources and examined whether they inhibited CT-
catalyzed ADP-ribosylation. The inhibitory activity of
non-catechin polyphenols such as chlorogenic acid,
phloridzin, phloretin, and p-coumaric acid at 25 µg/ml
was only weak: 12.7±5.5%, 4.6±3.8%, 8.4±6.6%,
2.8±5.0%, and 5.3±10.1% of the ADP-ribosylation
was inhibited, respectively. Monomeric catechins, such
as (－)-epicatechin and (＋)-catechin, did not inhibit it
Effect of APE on the Kinetics of ART Activity
To determine how APE inhibits the enzymatic activ-
ity of CT, a Lineweaver-Burk analysis was performed
(14). APE had no effect on the Km for NAD but signifi-
cantly reduced the maximal velocity (Fig. 4A). Unlike
the case with NAD, APE significantly increased the Km
for agmatine without altering the maximal velocity (Fig.
4B). These data suggest that APE had a different effect
on the ART activity of CT when agmatine or NAD was
examined. We concluded from these data that APE has
a negative allosteric effect on CT.
Discussion
In the present study, we demonstrated that APE, the
polyphenol extract from immature apples, inhibited CT-
catalyzed ADP-ribosylation and CT-induced fluid accu-
mulation in mice. It also turned out that the inhibitory
substances in the extract were highly polymerized cate-
chin compounds, although dimeric and trimeric cate-
chins also showed lower inhibitory activity. Although
these observations may simply suggest that APE inhibits
CT-induced fluid accumulation by disturbing the A sub-
unit-residing ADP-ribosyltransferase activity, the precise
mechanism for how polymerized catechins inactivate
the enzymatic activity of CT is not fully known at this
moment. Our preliminary observation, however, suggests
that the catechins tightly bind to CT, resulting in irre-

253
CHOLERA TOXIN INHIBITOR
supported by the observation that catechin compounds
from another plant source, which showed potent inhibi-
tion of the transferase activity, disturbed CT-induced
fluid accumulation to a much smaller degree than APE
(our unpublished observation). Future studies should
examine whether APE has the potential to act on intesti-
nal mucosal function.
The effective doses of APE to inhibit CT-induced
fluid accumulation in two bioassays differed considerably
(Figs. 2 and 3). The different effectiveness can be part-
ly explained by the difference in CT amounts used in the
assays; 40 times more CT was administered per mouse in
sealed mice model. It is considered that additional fac-
tors that caused the difference are route and timing of
administrations. In ileal loop assay, CT and APE were
injected simultaneously into the intestinal lumen where
the toxin acts. On the contrary, in sealed mice model the
toxin and APE were injected in the gastric tract, which is
generally filled with ingested food even after 48-hr star-
vation (our observation). The toxin and polyphenols
in gastric lumen should be then delivered to intestinal
tract, together with gastric and intestinal contents mixed
with digestive juice. Thus, the degradation and/or mod-
ification of APE might have occurred under the condi-
tion, resulting in the lower efficiency of APE to inactivate
CT in the sealed mice model.
Cholera patients are treated with an oral rehydration
solution, which relieves life-threatening dehydration and
reduces the mortality rate of the disease (16). Polyphe-
nols that inhibit CT-induced fluid accumulation may
exert an additional anti-diarrheal effect. For this purpose,
other investigators have reported that tea polyphenol
has an inhibitory effect on CT-induced fluid accumulation
in sealed adult mice and the ligated intestinal loops of
rabbits (19) and that it has a protective effect on gnoto-
biotic mice infected with the Escherichia coli O157:H7
strain (8). Similar to tea polyphenols, APE is frequent-
ly used as a food additive. Therefore, simultaneous
ingestion of APE with the oral rehydration solution
would be expected to reduce the CT-induced fluid accu-
mulation and protect intestinal mucosa from a secretory
response. The minimal APE concentration to inhibit
more than 90% of CT-catalyzed ADP-ribosylation was
estimated to be 25 µg/ml (Fig. 1). From a previous
report that describes the detection of CT in stools of
cholera patients (15), we conjectured that the concen-
tration of CT in the intestine is between 26 pg/ml and 100
ng/ml. Considering these data, more than 200 ng/ml of
APE would inactivate CT in the stools of the cholera
patients. It has been reported that stool excretion in
cholera patients is generally less than 10 liters/day but
reaches 30 liters/day in severe cases (12, 20). Thus, if a
cholera patient could take more than 30 mg of APE in a
Fig. 4. Effect of APE on the kinetics of ART activity. Assay
contained A: 15 µg/ml CT, 100 µM NAD, and the indicated con-
centrations of agmatine, B: 15 µg/ml CT, 20 mM agmatine, and
the indicated concentrations of NAD. (○) no additions; (●)
8.7 µg/ml APE.
versible inactivation of the toxin (our unpublished obser-
vation). An additional explanation for the in vivo effect
of APE is that APE not only inhibits the transferase
activity of CT but also modifies a function of the intesti-
nal mucosa, protecting it from a secretory response
against the diarrhegenic bacterial toxin. It has been
reported that proanthocyanidins affect the intestinal
mucosal function, resulting in a reduction of CT-induced
fluid accumulation (7). Since proanthocyanidins are
categorized from their structure into the same group as
the catechin polymer in APE (22), it is probable that the
polymerized catechins in APE also show similar pro-
tective effects on the host intestine. Our hypothesis that
APE shows a protective effect on intestinal mucosa is

254
T. SAITO ET AL
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gut lumen at a concentration of more than 1 µg/ml, the
APE might neutralize CT, resulting in the reduction of
water excretion from the patient’s bowel. APE itself is
not toxic to humans and can be administered without
apparent side effects at a dose of 10 mg/kg/day (10).
Considering all the above-mentioned results, we think
that APE could be used on cholera patients as a precau-
tionary or therapeutic agent. Further studies with cholera
patients are necessary in order to confirm the efficacy of
APE for these purposes.
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Although APE was similar to the tea polyphenols in
that both polyphenol preparations exhibited a protec-
tive effect against the diarrhegenic activity of CT, the dif-
ference in their polyphenol compositions should be
noted. Toda and her collaborators, who have reported the
inhibitory activity of tea polyphenol on CT-induced fluid
accumulation in mice and rabbits, described the polyphe-
nol composition of their tea catechin preparation as fol-
lows (19): (－)-epigallocatechin gallate (59%, dry
weight/dry weight), (－)-epigallocatechin (19%), (－)-
epicatechin gallate (14%), (－)-epicatechin (6%), and
(＋)-gallocatechin (2%). This suggests that monomeric
catechins and their galloyl derivatives would be the
major inhibitory substances in tea polyphenols. Unlike
the tea polyphenols, the major inhibitory polyphenols in
APE were considered to be polymerized catechins in this
study. Therefore, it is of interest how these structurally
unrelated polyphenols show inhibitory activity on the
same bacterial toxin. Future studies may shed light on
the structure-function relationship of the inhibitors for
bacterial ADP-ribosyltransferase.
8) Isogai, E., Isogai, H., Takeshi, K., and Nishikawa, T. 1998.
Protective effect of Japanese green tea extract on gnotobiotic
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Toyoda, M., Teshima, R., and Saito, Y. 1998. Inhibitory
effects of apple polyphenol on induced histamine release
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Toyoda, M., and Kobayashi, Y. 2000. Anti-allergic effect of
apple polyphenol on patients with atopic dermatitis: a pilot
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R.A. 1968. Oral maintenance therapy for cholera in adults.
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We thank Dr. Iwao Kato and Dr. Yoshifumi Takeda for their
participation in helpful discussions. We are grateful to Dr. Jyunei
Kinjo for the fraction of APE into four FAPs. In addition, we
thank Ms. Emi Utsuno and Ms. Michiko Hatano for their technical
assistance.
This work was supported by Grants-in-Aid of Scientific
Research (03454180, 04304032, 05271205, 06264204, and
08770184) from the Ministry of Education of Japan and Grants
for International Health Cooperation Research (11A-1-1999,
11A-1-2000) from the Ministry of Health and Welfare, Japan.
Acta 1034: 195―199.
15) Ramamurthy, T., Bhattacharya, S.K., Uesaki, Y., Horigome,
K., Paul, M., Sen, D., Pal, S.C., Takeda, T., Takeda, Y., and
Nair, G.B. 1992. Evaluation of the bead enzyme-linked
immunosorbent assay for detection of cholera toxin directly
from stool specimens. J. Clin. Microbiol. 30: 1783
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