Ginger and its bioactive component inhibit enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea in mice

Article abstract of DOI:10.1021/jf071460f

Ginger is one of the most commonly used fresh herbs and spices. Enterotoxigenic Escherichia coli heat-labile enterotoxin (LT)-induced diarrhea is the leading cause of infant death in developing countries. In this study, we demonstrated that ginger significantly blocked the binding of LT to cell-surface receptor G_M1, resulting in the inhibition of fluid accumulation in the closed ileal loops of mice. Biological-activity-guided searching for active components showed that zingerone (vanillylacetone) was the likely active constituent responsible for the antidiarrheal efficacy of ginger. Further analysis of chemically synthesized zingerone derivatives revealed that compound 31 (2-[(4-methoxybenzyl)oxy]benzoic acid) significantly suppressed LT-induced diarrhea in mice via an excellent surface complementarity with the B subunits of LT. In conclusion, our findings provide evidence that ginger and its derivatives may be effective herbal supplements for the clinical treatment of enterotoxigenic Escherichia coli diarrhea.

Full text of DOI:10.1021/jf071460f

8390 J. Agric. Food Chem. 2007, 55, 8390–8397
Ginger and Its Bioactive Component Inhibit
Enterotoxigenic Escherichia coli Heat-Labile
Enterotoxin-Induced Diarrhea in Mice
JAW-CHYUN CHEN,† LI-JIAU HUANG,‡ SHIH-LU WU,§ SHENG-CHU KUO,‡
TIN-YUN HO,*,| AND CHIEN-YUN HSIANG*,
Graduate Institute of Chinese Pharmaceutical Sciences, Graduate Institute of Pharmaceutical
Chemistry, Department of Biochemistry, Graduate Institute of Chinese Medical Science, Department
of Microbiology, China Medical University, Taichung 40402, Taiwan
Ginger is one of the most commonly used fresh herbs and spices. Enterotoxigenic Escherichia coli heat-
labile enterotoxin (LT)-induced diarrhea is the leading cause of infant death in developing countries. In
this study, we demonstrated that ginger significantly blocked the binding of LT to cell-surface receptor
GM1, resulting in the inhibition of fluid accumulation in the closed ileal loops of mice. Biological-activity-
guided searching for active components showed that zingerone (vanillylacetone) was the likely active
constituent responsible for the antidiarrheal efficacy of ginger. Further analysis of chemically synthesized
zingerone derivatives revealed that compound 31 (2-[(4-methoxybenzyl)oxy]benzoic acid) significantly
suppressed LT-induced diarrhea in mice via an excellent surface complementarity with the B subunits of
LT. In conclusion, our findings provide evidence that ginger and its derivatives may be effective herbal
supplements for the clinical treatment of enterotoxigenic Escherichia coli diarrhea.
KEYWORDS: Ginger; zingerone; enterotoxigenic Escherichia coli; heat-labile enterotoxin; diarrhea
INTRODUCTION
Escherichia coli (ETEC) is the leading bacterial cause of
pediatric diarrhea, accounting for 210 million diarrhea episodes
and approximately 380000 deaths annually. (5, 6) Heat-labile
enterotoxin (LT) is the major virulence factor of ETEC (7). LT
consists of one A subunit for catalytic activity and five B
subunits for receptor binding (8, 9). When the B subunit (LTB)
binds to the ganglioside GM1 on the intestinal cell surface, it
induces a conformational change in the toxin molecule, followed
by the translocation of the A subunit (LTA) into the cells. Inside
the intestinal cells, LTA catalyzes the ADP-ribosylation of the
stimulatory GTP-binding protein, resulting in the increased
intracellular levels of cyclic AMP. The elevated levels of cyclic
AMP in the cells cause massive losses of fluid and ions from
the cells, leading to the symptom of diarrhea (10). Because the
binding of LTB to GM1 is the first step of LT-induced diarrhea,
it is an attractive target for developing drugs for the treatment
and prophylaxis of LT-induced diarrhea (9).
Ginger, the rhizome of Zingiber officinale, is a common
condiment for various foods and beverages. Ginger has a long
history of medicinal use in China and India for the treatment
of headaches, nausea, rheumatism, and colds (1). Clinical studies
indicate that ginger can be used as a “broad-spectrum anti-
emetic”. It prevents nausea and/or emesis resulting from
pregnancy, postoperation, motion sickness, and other causes
(acetonemia or cytostatics) (2, 3). Pharmacological studies
indicate that ginger exerts anti-inflammatory, antipyretic, anti-
microbial, antischistosomal, antitumorigenic, antioxidative, hy-
poglycemic hepatoprotective, diuretic, and hypocholesterolemic
effects in Vitro (4). Furthermore, ginger exhibits wide effects
on the gastrointestinal tract by increasing bile secretion;
preventing the occurrence of gastric ulcers; and enhancing
pancreatic lipase, intestinal lipase, disaccharidases, sucrase, and
maltase activities in animal models (4).
Many medical plants and their constituents have been used
for the treatment of diarrhea. For examples, Croton lechleri,
Galla chinensis, rhubarb, and tea suppress enterotoxin-induced
diarrhea through various mechanisms (11–14). In this study,
we evaluated the antidiarrheal potential of ginger on the basis
of its inhibitory effect on the LTB and GM1 interaction. Our
data show that ginger was effective in the inhibition of LT-
induced diarrhea via blocking the LTB and GM1 interaction.
Zingerone was the likely active constituent of ginger responsible
for antidiarrheal activity. Analysis of chemically synthesized
derivatives of ginger constituents further suggested that com-
Diarrhea caused by intestinal pathogens is the leading cause
of infant death in developing countries (5). Enterotoxigenic
* To whom correspondence should be addressed. Tel.: +886 4
22053366ext. 2163 (C.-Y.H.); +886 4 22053366ext. 3302 (T.-Y.H.).
Fax: +886 4 22053764 (C.-Y.H.); +886 4 22032295 (T.-Y.H.). E-mail:
cyhsiang@mail.cmu.edu.tw (C.-Y.H.); tyh@mail.cmu.edu.tw (T.-Y.H.).
† Graduate Institute of Chinese Pharmaceutical Sciences.
‡ Graduate Institute of Pharmaceutical Chemistry.
§ Department of Biochemistry.
| Graduate Institute of Chinese Medical Science.
Department of Microbiology.
10.1021/jf071460f CCC: $37.00
2007 American Chemical Society
Published on Web 09/20/2007

Ginger Inhibits E. coli LT-Induced Diarrhea in Mice
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8391
previously (13). Briefly, mice were fasted for 24 h with water available
ad libitum. Mice were then anesthetized with ketamine and xylazine,
and the intestines were exteriorized through a midline incision. One
intestinal segment (about 4 cm) was ligated, and the LT (1 µg) and/or
compounds in a total volume of 200 µL were simultaneously injected
into the loop. Twenty-four hours later, the mice were sacrificed and
the loops were excised. The fluid accumulation (g/cm) was calculated
by dividing the weight of the fluid-containing loop by the length of
the loop.
Patent Mouse Gut Assay. LT-induced diarrhea was evaluated by
patent mouse gut assay as described previously (12). Briefly, six mice
per group were fasted for 16 h with water available ad libitum. Each
mouse was inoculated intragastrically with 0.5 mL of 10 µg LT and/or
compounds. Six hours later, the mice were sacrificed. The entire
intestine from duodenum to rectum was carefully removed to retain
any accumulated fluid, and the residual mesentery was removed prior
to weighing. The carcass was weighed separately. LT-induced diarrheal
ability was presented as the gut/carcass weight ratio by dividing the
weight of gut by the weight of the carcass.
Docking Analysis. Gasteiger–Huckel charges were deployed to
compounds in the Maybridge database using the SYBYL 7.0.1 program.
The position and conformation of each compound were optimized by
the anchor fragment orientation and then torsion minimization methods
implemented in the DOCK4.0.2 program or DOCK5.3.0 (18). There
were 50 configurations and 100 maximum anchor orientations for each
compound used in the anchor-first docking algorithm. All the docked
configurations were energy-minimized by 100 maximum iterations and
one minimization cycle. The X-Score program was used to correct the
docking score (19). The LIGPLOT 4.22 program was used to identify
specific contacts between ligands and LTB (20).
pound 31 (2-[(4-methoxybenzyl)oxy]benzoic acid) might be the
lead for further optimization.
MATERIALS AND METHODS
Extraction and Fractionation of Ginger. The dried ginger was a
gift from Sun Ten Pharmaceutical Corporation (Taipei, Taiwan). The
plant sample was ground with a homogenizer to a fine powder and
extracted by mixing 100 g of the herb powder with methanol at 4 °C
overnight. The supernatant was then collected and stored at –30 °C in
small aliquots. The ginger extract was further evaporated under a
vacuum to dryness, resuspended in deionized water, and partitioned
with four different solvents (n-hexane, chloroform, ethyl acetate, and
n-butanol) to yield n-hexane, chloroform, ethyl acetate, n-butanol, and
aqueous fractions, respectively. Each fraction was concentrated under
reduced pressure at a temperature less than 40 °C, and the solid mass
was then dissolved in methanol, divided into small aliquots, and kept
at –30 °C until use.
Preparation of Zingerone Derivatives. Zingerone, 6-gingerol, and
geraniol were purchased from Sigma (St. Louis, MO). 1-Phenyl-3,5-
dodecenediones and 3-phenyl-acrylaldehydes were synthesized as
described previously (15, 16). Briefly, 3,4-substituted cinnamic acids
were used as starting materials to synthesize gingerdiones and dehydro-
gingerdione derivatives (1-phenyl-3,5-dodecenediones). Cinnamic al-
dehyde was reacted with a variety of substituted benzyl chlorides to
synthesize 3-phenyl-acrylaldehydes. The benzyloxybenzenes were
synthesized as follows. The starting substituted phenols were subjected
to O-benzylation by reacting with substituted benzyl chloride in the
presence of K2CO3 and KI to yield the corresponding substituted
benzyloxybenzene derivatives (compounds 1–25). Substituted benz-
yloxybenzoates were allowed to react with sodium hydroxide to afford
corresponding substituted benzyloxybenzoic acids (compounds
26–34).
Statistical Analysis. Data were presented as mean ( standard errors.
Student’s t test was used for comparisons between two experiments.
A value of p < 0.05 was considered statistically significant.
Expression and Purification of Escherichia coli LT and LTB.
RecombinantLTandLTBwereexpressedintheE.coliBL21(DE3)pLysS
strain and purified by affinity chromatography as described previously
(12). Briefly, cells induced by isopropyl-ꢀ-D-thiogalactopyranoside were
collected 3 h after induction. The cell pellet was resuspended in TEAN
buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 200 mM NaCl, and 3
mM Na3N), lysed by sonication, and centrifuged at 15000g for 20 min
at 4 °C. The supernatant was collected and mixed with D-galactose
resin (Pierce, Rockford, IL), and the recombinant LT and LTB were
then eluted by 1× TEAN buffer containing 1 M galactose. Proteins
were analyzed by sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis and quantified with a Bradford assay (Bio-Rad, Hercules,
CA).
Biotinylation of LTB. LTB was biotinylated as described previously
(19). Briefly, LTB was mixed with Sulfo-NHS-LS-biotin (Pierce,
Rockford, IL) in a ratio of 1:10. After a 2 h incubation on ice, the
unincorporated biotin was removed by centricon-10 (Millipore, Bedford,
MA), and the biotinylated LTB was stored at 4 °C until further
analysis.
RESULTS
Effects of Ginger on the Binding of LTB to GM1. The
inhibitory abilities of ginger on the binding of LTB to GM1 were
evaluated by GM1-ELISA. As shown in Figure 1A, ginger
exhibited a significant effect on the abolishment of the LTB–GM1
interaction. The inhibitory effect of the ginger extract was dose-
dependent, with an IC50 value of 2.0 µg/mL. These data suggest
that ginger might have antidiarrheal activities by blocking the
binding of LTB to GM1
.
Effects of Ginger on LT-Induced Fluid Accumulation in
Mice. The antidiarrheal efficacy of ginger extract was tested in
ViVo using the toxin-induced fluid accumulation assay in the
short circuit of small intestines in mice. In this assay, the closed
loops of small intestines were created in ViVo, and the lumens
of loops were injected with small volumes of saline, LT, or
LT-containing ginger extracts. Luminal fluid accumulation was
determined after 24 h. As seen in Figure 1B, there was marked
fluid accumulation and distention in LT-treated loops, whereas
the normal (saline) loop remained empty. The methanolic extract
of ginger significantly suppressed fluid accumulation in the
toxin-treated intestinal loops in a dose-dependent manner.
Methanol alone did not affect the fluid accumulation induced
by LT. These findings indicate that the ginger extract blocks
the binding of LTB to GM1, resulting in the suppression of LT-
induced diarrhea.
GM1-Enzyme-Linked Immunosorbent Assay (ELISA). The inter-
action of LTB with ganglioside GM1 was evaluated by a GM1-ELISA
as described previously (12). Briefly, biotinylated LTB (16 ng) was
mixed with various amounts of the compound and incubated at 4 °C
with shaking. After a 3 h incubation, the mixture was added to wells,
which were coated with 200 ng of GM1, and incubated at 37 °C for
1 h. Following three washes, peroxidase-conjugated avidin and a
chromatic substrate, 2,2′-azinobis(3-ethylbenzithiazoline-sulfonic acid),
were sequentially added. The absorbance was read at 405 nm in an
ELISA plate reader (Anthos Labtec Instruments, Austria). The percent-
age of inhibition was calculated by [1 –(OD value of mixture containing
LTB and compound/OD value of mixture containing LTB only)] ×
100.
Fluid Accumulation Assay. Female BALB/c mice (8 weeks old,
20 ( 1 g weight) were obtained from the National Laboratory Animal
Center (Taipei, Taiwan). Mouse experiments were conducted under
ethics approval from the China Medical University Animal Ethics
Committee (Ethics Approval Number 2005-3). Fluid accumulation
induced by LT was evaluated with mouse ileal loops as described
Effects of Zingerone, a Ginger Constituent, on LT-
Induced Fluid Accumulation and LTB-GM1 Interaction.
Biological-activity-guided searching for active components was
performed by screening various solvent fractions of ginger using
GM1-ELISA. As shown in Figure 2, aqueous, n-butanol soluble,
and ethyl acetate soluble fractions of ginger did not inhibit the
interaction between LTB and GM1. However, the fractions from
nonpolar solvents (n-hexane and chloroform) blocked the

8392 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Chen et al.
Figure 2. Inhibitory effects of ginger fractions on the interaction of LTB
with GM1. Various amounts of ginger fractions were incubated with 16 ng
of biotinylated LTB, and GM1-ELISA was performed as described in the
Materials and Methods. Results are expressed as inhibition (%). Values
are mean ( standard errors of two independent assays.
Figure 1. Antidiarrheal effect of ginger. (A) GM1-ELISA. Various amounts
of ginger extract were incubated with 16 ng of biotinylated LTB, and GM1-
ELISA was performed as described in the Materials and Methods. Results
are expressed as inhibition (%). Values are mean ( standard errors of
six independent assays. (B) Fluid accumulation assay. LT (1 µg) and/or
various amounts of ginger extract were simultaneously injected into the
ileal loops, and mice were sacrificed 24 h later. Cross appearance of the
ileal loops is shown at the top. The dissected ileal loops from a normal
mouse treated with saline and a solvent control mouse treated with LT
and methanol are shown in the first and the second columns, respectively.
Fluid accumulation (g/cm) is shown at the bottom. Values are mean (
standard errors of three independent assays. *p < 0.05, compared with
LT treatment.
binding of LTB to GM1 in a dose-dependent manner. These data
indicate that the active constituents of ginger might be lipophilic.
Zingerone (vanillylacetone), 6-gingerol, and geraniol, which are
present in the ginger oil (21–23), were further tested for their
inhibitory effects. As shown in Figure 3B, zingerone and
Figure 3. Inhibitory effects of zingerone, 6-gingerol, and geraniol on the
LTB–GM1 interaction and LT-induced fluid accumulation. (A) Structures
of zingerone, 6-gingerol, and geraniol. (B) GM1-ELISA. Various amounts
of zingerone, 6-gingerol, and geraniol were incubated with 16 ng of
biotinylated LTB. Results are expressed as inhibition (%). Values are mean
( standard errors of two independent assays. (C) Fluid accumulation
assay. LT (1 µg) and/or various amounts of zingerone were simultaneously
injected into the ileal loops, and mice were sacrificed 24 h later. Results
are expressed as fluid accumulation (g/cm). Values are mean ( standard
errors of three independent assays. *p < 0.05, **p < 0.01, compared with
LT treatment.
6-gingerol significantly abolished the binding of LTB to GM1
,
while geraniol did not affect the LTB–GM1 interaction. Antidi-
arrheal efficacies of zingerone and 6-gingerol were further
evaluated by a fluid accumulation assay. Zingerone significantly
inhibited LT-induced fluid accumulation in the closed loops
(Figure 3C), while 6-gingerol did not affect it (data not shown).
The IC50 of zingerone was 2.5 mM. These findings indicate that
zingerone was the likely active constituent responsible for the
antidiarrheal activity of ginger.
and 6-gingerol, we found that they shared a common skeleton
with a benzene ring and a hydrocarbon chain. We wondered
whether the number of carbon atoms or phenolic groups was
Effect of Compound 31, a Zingerone Derivative, on the
Binding of LTB to GM1. Comparing the structures of zingerone

Ginger Inhibits E. coli LT-Induced Diarrhea in Mice
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8393
Figure 4. Inhibitory effects of zingerone derivatives on the LTB and GM1 interaction. (A) Structures of 1-phenyl-3,5-dodecenedione, 1-phenyl-acrylaldehyde,
and benzyloxybenzene. (B) Solution-phase synthesis of benzyloxybenzoic acid analogs. (C) GM1-ELISA. Biotinylated LTB (16 ng) was mixed with 10 mM
benzyloxybenzoic acid analogs. Results are expressed as inhibition (%). Values are mean ( standard errors of two independent assays.
significant for the interaction with LTB; various derivatives were
synthesized and their inhibitory effects on the LTB-GM1
interaction were evaluated by GM1-ELISA (Figure 4). We first
elongated and shortened the carbon chain to synthesize 1-phenyl-
3,5-dodecenediones and 3-phenyl-acrylaldehydes, respectively
(Figure 4A). Unfortunately, these compounds showed no effect
on the LTB and GM1 interaction (data not shown). We further
combined two phenolic groups to synthesize benzyloxybenzene
derivatives (Figure 4B and Table 1). Data showed that
benzyloxybenzene derivatives displayed various inhibitory ef-
ficacies on the binding of LTB to GM1 (Figure 4C). Compounds
8 and 33 exhibited approximately 50% inhibition, while
compound 31 (2-[(4-methoxybenzyl)oxy]benzoic acid) displayed
a 90% inhibition on the LTB–GM1 interaction. In comparison
with m-nitrophenyl-R-D-galactopyranoside (MNPG), the recep-
tor-binding antagonist derived from the simple sugar galactose
(24), we found that compound 31 displayed a more effective
inhibition (Figure 5). At 10 mM, compound 31 exhibited a 90%
inhibition, while MNPG displayed an approximately 20%
inhibition on the LTB and GM1 interaction.
Effect of Compound 31 on LT-Induced Diarrhea in Mice.
Fluid accumulation and patent mouse gut assay were performed
to evaluate the antidiarrheal effect of compound 31. As shown
in Figure 6A, LT induced massive fluid accumulation in the
closed ileal loops. Solvent (dimethyl sulfoxide, DMSO) alone
did not decrease the volume of LT-induced fluid in the intestinal
loops. However, compound 31 suppressed LT-induced intestinal
fluid secretion in a dose-dependent manner, with the IC50 of
0.1 mM. The patent mouse gut assay also showed that
compound 31 significantly inhibited LT-induced fluid ac-
cumulation in the gut, with a concentration-dependent decrease
in the gut–carcass ratio (Figure 6B). These findings indicate
that compound 31, a zingerone derivative, exhibited antidiarrheal
effects by blocking the LTB–GM1 interaction. Moreover, it was
more effective than zingerone in the prevention of LT-induced
diarrhea.
Compound 31 Interacted with LTB. DOCK and X-Score
programs were performed to analyze the interaction between
compound 31 and LTB (see the Supporting Information for
results). The docking data of MNPG were consistent with the
X-ray diffraction data, in which there are hydrogen-bond
interactions with the side chains of Gly-33, Gln-61, and Asn-
90, and hydrophobic contacts with the side chains of His-57
and Trp-88 of LTB (Figure 7A) (25). Compound 31 docked
into LTB with complementarity (Figure 7A). Critical hydrogen-
bond interactions with the side chains of Arg-13 and Asn-90
and hydrophobic contacts with the side chains of Tyr-12, His-
57, and Trp-88 of LTB were found in the LTB and compound
31 interactions. Comparing the docking results of MNPG and
compound 31, we found that they shared a hydrogen-bond
interaction with Asn-90 and two hydrophobic interactions with
His-57 and Trp-88 of LTB (Figure 7B). Analysis of the binding
affinity of LTB–compound interaction showed that the docking
score of compound 31 was higher than that of MNPG. These
findings suggest that compound 31 may be more effective than
MNPG in binding to LTB.

8394 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Chen et al.
Table 1. Functional Groups of 34 Benzyloxybenzene Derivatives
group
1
compound
R1
CN
CN
CN
CN
CN
R2′
R3′
R4′
1
2
3
4
H
Cl
H
H
F
H
H
Cl
H
H
H
H
H
Cl
H
5
6
7
8
9
CN
CN
CN
CN
CH3
CH3
CH3
CH3
H
H
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
H
F
F
H
OCH3
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
F
H
OCH3
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
2
3
4
5
6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
CH2OH
CH2OH
CH2OH
CH2OH
COOCH3
COOCH3
COOCH3
COOCH3
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
H
Cl
H
OCH3
H
H
H
H
Cl
H
OCH3
H
H
H
F
H
H
F
Figure 6. Antidiarrheal effect of compound 31 on the LT-induced diarrhea
in mice. (A) Fluid accumulation assay. LT (1 µg) and/or various amounts
of compound 31 were simultaneously injected into the ileal loops, and
mice were sacrificed 24 h later. Cross appearance of the ileal loops is
shown at the top. The dissected ileal loops from a normal mouse treated
with saline and a solvent control mouse treated with LT and DMSO are
shown in the first and the second columns, respectively. Fluid accumulation
(g/cm) is shown at the bottom. (B) Patent mouse gut assay. Six mice per
group were orally administered saline, 10 µg of LT, 10 µg of LT mixed
with DMSO, or 10 µg of LT mixed with various amounts of compound
31. After a 6 h incubation, the mice were sacrificed. The gut/carcass
weight ratio was calculated for each group. Values are mean ( standard
errors of triplicate assays. *p < 0.05, compared with LT treatment.
Figure 5. Inhibitory effects of MNPG and compound 31 on the binding
of LTB to GM1. Various amounts of MNPG, compound 31, or a solvent
(DMSO) were mixed with 16 ng of biotinylated LTB. Results are expressed
as inhibition (%). Values are mean ( standard errors of two independent
assays.
Ginger is one of the most commonly used fresh herbs and
spices. Ginger is among the 20 top-selling herbal supplements
in the United States (28). Ginger also ranks second in the top-
selling fresh herbs and spices in Australia, with an estimated
annual consumption of over 550 tons (24). Today, pharmaco-
poeias from different countries list that ginger extract is used
for various digestive diseases (29, 30). In this study, we
demonstrated for the first time that, in addition to an antiemetic
effect, ginger extracts exhibit antidiarrheal activities by blocking
the binding of LTB to GM1. Because LT and cholera toxin share
an 83% amino acid sequence homology, consisting of an A
subunit for enzymatic activity and five B subunits for receptor
recognition, and the fact that LT causes choleralike diarrhea
(10), our findings suggest that ginger extract could be used as
an herbal supplement to prevent choleralike diarrhea in develop-
ing countries.
DISCUSSION
Today, the most common principle of management for
diarrhea is fluid replacement combined with pharmacologic
therapy (26). Fluid and electrolyte replacement is used to replace
fluid losses by the administration of sugar–electrolyte solutions;
however, it does not facilitate the reabsorption of secreted fluid
and therefore does not lessen diarrhea. Antibiotics and antimo-
tility agents are used to control symptoms. Antibiotics kill
bacteria but cannot inhibit the toxicity of the bacterial toxin.
Moreover, antibiotic therapy is not a viable solution because
of the rapid increase in antibiotic resistance (27). Antimotility
agents, such as loperamide and diphenoxylate, can lessen stool
frequency and volume in mild diarrheas. However, such agents
are not suitable in severe diarrhea because they may cause
pooling of large fluid volumes in paralyzed bowel loops.
The LT holotoxin structure points to three potential target
areas for inhibitor design: blocking the enzyme-active site
located in the A1 domain, inhibiting holotoxin assembly by

Ginger Inhibits E. coli LT-Induced Diarrhea in Mice
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8395
Figure 7. Interaction of compound 31 with the receptor-binding site of LTB. (A) Schematic illustrations of the interactions of MNPG (top panel) and
compound 31 (bottom panel) with residues around the receptor-binding site of LTB. (B) Superimposed structure of MNPG (purple) and compound 31
(green) bound to LTB.
interrupting A2-B pentamer interactions, and blocking the toxin
and cellular receptor interaction (8, 9). We screened the
antidiarrheal potential of herbs on the basis of the LTB–GM1
interaction. Their antidiarrheal abilities were further evaluated
by a fluid accumulation assay. Results showed that there were
correlations between the inhibition of the LTB–GM1 interaction
and the suppression of LT-induced diarrhea in most compounds
(data not shown). One exception was 6-gingerol, which dis-
played an inhibitory effect on the LTB–GM1 interaction, while
it did not inhibit LT-induced diarrhea in mice. Nakazawa and
Ohsawa (31) indicated that 6-gingerol is metabolized by gut
flora to various metabolites, such as vanillin acid, ferulic acid,
and 6-gingerol-4-O-ꢀ-glucuronide. The metabolic fate of 6-gin-
gerol by normal flora in the gut might explain why it failed in
the fluid accumulation assay.
Zingerone was the likely active constituent responsible for
the antidiarrheal activity of ginger. Three series of zingerone
derivatives were synthesized, and data showed that compound
31 (2-[(4-methoxybenzyl)oxy]benzoic acid) was more active
than zingerone both in Vitro and in ViVo. Additionally, the in

8396 J. Agric. Food Chem., Vol. 55, No. 21, 2007
Chen et al.
(4) Chrubasik, S.; Pittler, M. H.; Roufogalis, B. D. Zingiberis rhizoma:
a comprehensive review on the ginger effect and efficacy profiles.
Phytomedicine 2005, 12, 684–701.
(5) Kosek, M.; Bern, C.; Guerrant, R. L. The global burden of
diarrhoeal disease, as estimated from studies published between
1992 and 2000. Bull. W. H. O. 2003, 81, 197–204.
Vitro assay indicated that compound 31 was more effective than
MNPG in the inhibition of the LTB–GM1 interaction. Like
MNPG, compound 31 interacted with the surface of LTB via
hydrogen bonds and hydrophobic contacts. The phenolic sub-
stituents of compound 31 and MNPG increased the area of
hydrophobic contact with the toxin. In contrast, MNPG buried
roughly half of the binding-site surface covered by the full
receptor GM1 pentasaccharide (32), while compound 31 buried
the binding-site surface and extruded into the surface. These
findings suggested that compound 31 might form a stereobarrier
to exclude the entry of GM1. Because compound 31 significantly
suppressed the LT-induced diarrhea in mice and no detectable
tissue damage was observed (data not shown), compound 31
might be the lead candidate for further optimization.
(6) Nataro, J. P.; Kaper, J. B. Diarrheagenic Escherichia coli. Clin.
Microbiol. ReV. 1998, 11, 142–201.
(7) Evans, D. G.; Evans, D. J.; DuPont, H. L. Virulence factors of
enterotoxigenic Escherichia coli. J. Infect. Dis. 1997, 136 (Suppl.),
118–123 .
(8) Sixma, T. K.; Pronk, S. E.; Kalk, K. H.; Wartna, E. S.; van Zanten,
B. A.; Witholt, B.; Hol, W. G. Crystal structure of a cholera toxin-
related heat labile enterotoxin from E. coli. Nature 1991, 351,
371–377.
In conclusion, pharmacologic therapy specifically targeting
the bacterial toxin is an ideal management for the treatment or
prevention of diarrhea. However, today, the most common
principle of management for diarrhea is either the replacement
of fluid losses (fluid replacement therapy) or the killing of
bacteria (antibiotics therapy). In this study, we demonstrated
that ginger, the commonly used herb and spice, was effective
in inhibiting choleralike diarrhea in mice via the abolishment
of toxin–receptor interaction. By biological-activity-guided
searching for active components of ginger, zingerone was
identified as the likely active constituent responsible for the
observed antidiarrheal activity of ginger. Further analysis of the
chemically synthesized zingerone derivatives revealed that
compound 31 was more effective than zingerone in both the
blockade of toxin-receptor interaction and the suppression of
diarrhea in mice. The U.S. Food and Drug Administration
classifies ginger as “Generally Recognized as Safe,” and the
German Commission E Monographs report that ginger has no
known side effects and no known drug/herb interactions (30).
Therefore, our observations suggest that ginger may be an
effective candidate for the clinical treatment of ETEC diarrhea.
Furthermore, our findings demonstrate for the first time that LT
receptor-binding antagonists are effective in the suppression of
LT-induced diarrhea in ViVo.
(9) Sixma, T. K.; Pronk, S. E.; Kalk, K. H.; van Zanten, B. A.;
Berghuis, A. M.; Hol, W. G. Lactose binding to heat-labile
enterotoxin revealed by X-ray crystallography. Nature 1992, 355,
561–564.
(10) Spangler, B. D. Structure and function of cholera toxin and the
related Escherichia coli heat-labile enterotoxin. Microbiol. ReV.
1992, 56, 622–647.
(11) Fischer, H.; Machen, T. E.; Widdicombe, J. H.; Carlson, T. J.;
King, S. R.; Chow, J. W.; Illek, B. A novel extract SB-300 from
the stem bark latex of Croton lechleri inhibits CFTR-mediated
chloride secretion in human colonic epithelial cells. J. Ethnop-
harmacol. 2004, 93, 351–357.
(12) Chen, J. C.; Ho, T. Y.; Chang, Y. S.; Wu, S. L.; Hsiang, C. Y.
Anti-diarrheal effect of Galla Chinensis on the Escherichia coli
heat-labile enterotoxin and ganglioside interaction. J. Ethnophar-
macol. 2006, 103, 385–391.
(13) Oi, H.; Matsuura, D.; Miyake, M.; Ueno, M.; Takai, I.; Yamamoto,
T.; Kubo, M.; Moss, J.; Noda, M. Identification in traditional
herbal medications and confirmation by synthesis of factors that
inhibit cholera toxin-induced fluid accumulation. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 3042–3046.
(14) Bruins, M. J.; Cermak, R.; Kiers, J. L.; van der Meulen, J.; van
Amelsvoort, J. M.; van Klinken, B. J. In ViVo and in Vitro effects
of tea extracts on enterotoxigenic Escherichia coli-induced
intestinal fluid loss in animal models. J. Pediatr. Gastroenterol.
Nutr. 2006, 43, 459–469.
(15) Hsu, M. H.; Kuo, S. C.; Chen, C. J.; Chung, J. G.; Lai, Y. Y.;
Huang, L. J. 1-(3,4-Dimethoxyphenyl)-3,5-dodecenedione (I6)
induces G1 arrest and apoptosis in human promyelocytic leukemia
HL-60 cells. Leuk. Res. 2005, 29, 1399–1406.
(16) Huang, L. J.; Wang, J. P.; Lai, Y. C.; Kuo, S. C. Synthesis of
(2E)-3-{2-[(substituted benzyl)oxy]phenyl}acrylaldehydes as novel
anti-inflammatory agents. Bioorg. Med. Chem. Lett. 2006, 16,
2742–2747.
ABBREVIATIONS USED
ETEC, enterotoxigenic Escherichia coli; LT, heat-labile
enterotoxin; LTB, B subunit of LT; LTA, A subunit of LT; E.
coli, Escherichia coli; ELISA, enzyme-linked immunosorbent
assay; MNPG, m-nitrophenyl-R-D-galactopyranoside; DMSO,
dimethyl sulfoxide.
(17) Ho, T. Y.; Wu, S. L.; Chen, J. C.; Li, C. C.; Hsiang, C. Y. Emodin
blocks the SARS coronavirus spike protein and angiotensin-
converting enzyme 2 interaction. AntiViral Res. 2007, 74, 92–
101.
(18) Ewing, T. J. A.; Kuntz, I. D. Critical evaluation of search
algorithms for automated molecular docking and database screen-
ing. J. Comput. Chem. 1997, 18, 1175–1189.
(19) Wang, R.; Lai, L.; Wang, S. Further development and validation
of empirical scoring functions for structure-based binding affinity
prediction. J. Comput.-Aided Mol. Des. 2002, 16, 11–26.
(20) Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. LIGPLOT: a
program to generate schematic diagrams of protein-ligand interac-
tions. Protein Eng. 1995, 8, 127–134.
Supporting Information Available: Solid-phase synthesis of
1-phenyl-3,5-dodecenediones and 3-phenyl-acrylaldehydes, the
superimposition of cocrystal and docking structures of MNPG
around the receptor-binding domain of LTB, X-Score results
of MNPG and compound 31, and spectral characteristics of
benzyloxybenzene compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
(1) Langner, E.; Greifenberg, S.; Gruenwald, J. Ginger: history and
use. AdV. Ther. 1998, 15, 25–44.
(21) Connell, D. W.; Sutherland, M. D. A re-examination of gingerol,
shogaol, and zingerone, the pungent principles of ginger (Zingiber
officinale Roscoe). Aust. J. Chem. 1969, 22, 1033–1043.
(22) Jolad, S. D.; Lantz, R. C.; Solyom, A. M.; Chen, G. J.; Bates,
R. B.; Timmermann, B. N. Fresh organically grown ginger
(Zingiber officinale): composition and effects on LPS-induced
PGE2 production. Phytochemistry 2004, 65, 1937–1954.
(2) Ernst, E.; Pittler, M. H. Efficacy of ginger for nausea and vomiting:
a systematic review of randomized clinical trials. Br. J. Anaesth.
2000, 84, 367–371.
(3) Chaiyakunapruk, N.; Kitikannakorn, N.; Nathisuwan, S.; Leepra-
kobboon, K.; Leelasettagool, C. The efficacy of ginger for the
prevention of postoperative nausea and vomiting: a meta-analysis.
Am. J. Obstet. Gynecol. 2006, 194, 95–99.

Ginger Inhibits E. coli LT-Induced Diarrhea in Mice
J. Agric. Food Chem., Vol. 55, No. 21, 2007 8397
(23) Jolad, S. D.; Lantz, R. C.; Chen, G. J.; Bates, R. B.; Timmermann,
B. N. Commercially processed dry ginger (Zingiber officinale):
Composition and effects on LPS-stimulated PGE2 production.
Phytochemistry 2005, 66, 1614–1635.
(24) Parker, J. Culinary herbs. In The New Crop Industries Handbook;
Salvin, S., Bourke, M., Byrne, T., Eds; Rural Industries Research
and Development Corporation: Canberra, Australia, 2004; pp 236–
243.
(28) Blumenthal, M. Herb sales down 7.4 percent in mainstream
market. HerbalGram 2005, 66, 63.
(29) ESCOP Monograph. Zingiberis rhizome (Ginger); European
Scientific Cooperative on Phytotherapy: Elburg, Netherlands,
1996.
(30) Blumenthal, M. The Complete German Commission E Mono-
graphs: Therapeutic Guide to Herbal Medicines; Churchill
Livingstome: Oxford, England, 1998.
(25) Bernardi, A.; Raimondi, L.; Zuccotto, F. Simulation of protein-
sugar interactions: a computational model of the complex between
ganglioside GM1 and the heat-labile enterotoxin of Escherichia
coli. J. Med. Chem. 1997, 40, 1855–1862.
(26) Ahlquist, D. A.; Camilleri, M. Diarrhea and constipation. In
Harrison’s Principles of Internal Medicine, 15th ed.; Braunwald,
E., Fauci, A. S., Kasper, D. L., Hauser, S. L., Longo, D. L.,
Jameson, J. L., Eds.: McGraw-Hill: New York, 2001; pp 241–
250.
(27) Garg, P.; Chakraborty, S.; Basu, I.; Datta, S.; Rajendran, K.;
Bhattacharya, T.; Yamasaki, S.; Bhattacharya, S. K.; Takeda, Y.;
Nair, G. B.; Ramamurthy, T. Expanding multiple antibiotic
resistance among clinical strains ofVibrio cholerae isolated from
1992–1997 in Calcutta, India. Epidemiol. Infect. 2000, 124, 393–
399.
(31) Nakazawa, T.; Ohsawa, K. Metabolism of [6]-gingerol in rats.
Life Sci. 2002, 70, 2165–2175.
(32) Merritt, E. A.; Sarfaty, S.; Feil, I. K.; Hol, W. G. Structural
foundation for the design of receptor antagonists targeting
Escherichia coli heat-labile enterotoxin. Structure 1997, 5, 1485–
1499.
Received for review May 17, 2007. Revised manuscript received July
19, 2007. Accepted July 25, 2007. This work was supported by grants
from National Science Council (NSC 93-2320-B-039-029, NSC 95-2320-
B-039-004, and NSC 95-2320-B-039-027), Committee on Chinese
Medicine and Pharmacy (CCMP 96-RD-201, and China Medical
University (CMU95-053, CMU95-055, and CMU95-067).
JF071460F