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Helicobacter pylori is a Gram-negative, helical rod

that colonizes human gastric epithelium (45). In humans,


H. pylori plays a causal role in chronic gastritis and
peptic ulcer (5, 49), and is an important factor in the
occurrence of gastric cancer and gastric mucosa-associ-
ated lymphoid tissue lymphoma (MALToma) (4, 47).
Thus, the eradication of H. pylori can contribute to the
treatment and prevention of these diseases (10). H.
pylori eradication accelerates peptic ulcer healing (31),
reduces the recurrence of gastric cancer after resection
(42), and leads to regression of low-grade gastric MAL-
Toma (48). Currently, new triple therapies consisting of
two antibiotics and a proton pump inhibitor show high
eradication rates (43). However, some problems remain.
H. pylori rapidly acquires resistance to some antibi-
otics. H. pylori strains resistant to clarithromycin and
metronidazole are now increasing (35), which will reduce
eradication rates. In the near future, antibiotic resis-
tance will be the greatest obstacle in the treatment of H.
pylori infection. Furthermore, new triple therapies upon
occasion cause side effects; nausea, vomiting, epigastric
pain, abdominal discomfort, and diarrhea (46). There-
fore, a new antibacterial agent, which is both highly
effective and safe, is required for the treatment of H.
pylori infection.
It has been reported that various medicinal plant
Antibacterial Activity of Hydrolyzable Tannins
Derived from Medicinal Plants against
Helicobacter pylori
Keiji Funatogawa
1
, Shunji Hayashi*
, 1
, Hirofumi Shimomura
1
, Takashi Yoshida
2
,
Tsutomu Hatano
2
, Hideyuki Ito
2
, and Yoshikazu Hirai
1
1
Division of Bacteriology, Department of Infection and Immunity, Jichi Medical School, Tochigi 3290498, Japan, and
2
Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Okayama
7008530, Japan
Received September 17, 2003; in revised form, January 19, 2004. Accepted January 21, 2004
Abstract: Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we iso-
lated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity
against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity
against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds
demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tel-
limagrandin I demonstrated time- and dose-dependent bactericidal activity against H. pylori in vitro. On
the other hand, hydrolyzable tannins did not affect the viability of MKN-28 cells derived from human gas-
tric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against
H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes.
All the hydrolyzable tannins tested demonstrated dose-dependent membrane-damaging activity. Howev-
er, it remains to be elucidated whether their membrane-damaging activity directly contributes to their
antibacterial action.
Key words: Helicobacter pylori, Hydrolyzable tannins, Antibacterial activity, Membrane-damaging
activity
251
Microbiol. Immunol., 48(4), 251261, 2004
Abbreviations: CAG, cholesteryl-6-O-tetradecanoyl--D-glu-
copyranoside; CF, carboxyuorescein; CFU, colony-forming
unit; CG, cholesteryl glucoside; CGL, cholesteryl--D-glucopy-
ranoside; CPG, cholesteryl-6-O-phosphatidyl--D-glucopyran-
oside; DMCD, dimethyl--cyclodextrin; EC, Epicatechin; ECG,
Epicatechin gallate; EGCG, Epigallocatechin gallate; MAL-
Toma, mucosa-associated lymphoid tissue lymphoma; MIC,
minimum inhibitory concentration; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl-tetrazolium bromide; PBS, phosphate-buffered
saline; PC, phosphatidylcholin; RPMI, RPMI-1640 medium;
TG-I, Tellimagrandin I; TG-II, Tellimagrandin II.
*Address correspondence to Dr. Shunji Hayashi, Division of
Bacteriology, Department of Infection and Immunity, Jichi Med-
ical School, 33111 Yakushiji, Minamikawachi, Tochigi 329
0498, Japan. Fax: 81285441175. E-mail: shunhaya@jichi.
ac.jp
extracts have antibacterial activity (3, 1416). Thus,
these plants may contain new antibacterial agents that
have potential as new therapeutic agents against H.
pylori infection. In this study, we investigated antibac-
terial activity of compounds derived from medicinal
plants against H. pylori.
Materials and Methods
Bacterial strains. In this study, two standard strains
(NCTC 11638 and ATCC 43504) and 30 clinical isolates
of H. pylori were used. The clinical isolates were
obtained from nine patients with chronic gastritis, ten
patients with gastric ulcer and eleven patients with duo-
denal ulcer, and their identication was based on standard
biochemical tests (22). Stock cultures were stored at
80 C in brain heart infusion broth (Difco, Detroit,
Mich., U.S.A.) supplemented with 10% horse serum
and 10% glycerol. Subsequent analyses were performed
with strains derived from the frozen stocks. H. pylori
strains were grown on PPLO agar (Difco) supplemented
with 0.2% dimethyl--cyclodextrin (DMCD; Teijin,
Tokyo) at 37 C for 2 days in a microaerobic atmosphere
(5% O
2
/10% CO
2
/85% N
2
) and were used for this study
(33). In addition, one Escherichia coli strain (JC-2)
was cultured under the same conditions overnight and
was used for this study.
Test compounds. We isolated 36 polyphenols (20
hydrolyzable tannins, 3 catechins, 6 proanthocyanidins,
and 7 other polyphenols) and 4 terpenoids from medic-
inal plants used for the treatment of gastric disorders in
Asian countries (Table 1), and investigated their antibac-
terial activity. Furthermore, we selected ve hydrolyzable
tannins (TG-I, TG-II, Geraniin, Agrimoniin, and
Oenothein A) and two catechins (EGCG and EC) from
the above compounds, and analyzed their antibacterial
activity in detail. Chemical structures of the seven
selected compounds are shown in Fig. 1 (13, 23, 25,
53).
Acid-treated compounds. We also investigated
antibacterial activity of two hydrolyzable tannins (TG-I
and TG-II) treated with acid. Each compound (1 mg)
was dissolved in 1 ml of articial gastric juice, 20 mM
HCl (pH 1.8), and the solution was incubated at 37 C for
2 hr (54). After incubation, it was diluted with 10 mM
phosphate-buffered saline (PBS; pH 7.4), and was used
for the evaluation of antibacterial activity.
Antibacterial activity. Antibacterial screening of
plant-derived compounds was carried out using four H.
pylori strains (two standard strains and two clinical iso-
lates) and one E. coli strain. Besides these strains, 28
clinical isolates of H. pylori were used for the evaluation
of the seven selected compounds and the two acid-treat-
ed compounds. Minimum inhibitory concentrations
(MICs) were determined by the standard agar dilution
method (34). The test strains were suspended in PPLO
broth (Difco) at a concentration of approximately 10
7
colony-forming unit (CFU)/ml. The bacterial suspen-
sions (5 l) were inoculated onto 0.2% DMCD-supple-
mented PPLO agar plates containing serial twofold dilu-
tions of each compound with a multipoint inoculator
(Sakuma Seisakusho, Tokyo), and the plates were incu-
bated at 37 C for 3 days in a microaerobic atmosphere.
MICs were dened as the lowest concentrations that
visibly inhibited bacterial growth.
Bactericidal activity. Bactericidal activity was deter-
mined by using an in vitro killing assay (25, 29, 37). A
bacterial suspension of H. pylori NCTC 11638 (100 l,
approximately 10
7
CFU/ml) was inoculated into 10 ml of
0.2% DMCD-supplemented PPLO broth alone or con-
taining TG-I (12.5 to 50 g/ml). The cultures were
incubated with shaking at 37 C for 24 hr in a microaer-
obic atmosphere. Samples (100 l) for viability mea-
surement were taken at various points of time. Viabili-
ty was measured by the plate colony count technique.
After serial 10-fold dilution with PPLO broth, 100 l of
each sample was plated onto PPLO agar supplemented
with 0.2% DMCD. Colonies were counted after 3 days
of incubation at 37 C in a microaerobic atmosphere.
Effect on morphology of H. pylori. H. pylori NCTC
11638 was inoculated into 0.2% DMCD-supplemented
PPLO broth containing TG-I (50 g/ml). The culture
was incubated with shaking at 37 C for 24 hr in a
microaerobic atmosphere. The same broth, inoculated
with no bacteria, was also incubated in the same way as
control. Samples for morphological study were taken at
various times, then unxed and unstained H. pylori cells
were observed using a differential interference contrast
microscope (AX80; Olympus, Tokyo).
Preparation of liposomes. Liposomes were prepared
from egg-yolk phosphatidylcholin (PC; Sigma, St. Louis,
Mo., U.S.A.) (13). PC (0.5 mol) was dissolved in
chloroform and placed in a 10-ml round-bottomed ask.
The solvent was removed under negative pressure using
a rotary evaporator (RE-47; Yamato, Tokyo), and a thin
lipid lm was formed on the ask wall. The lipid lm
was dispersed in 100 l of PBS containing 200 mM car-
boxyuorescein (CF; Sigma) by agitation with a vortex
mixer (G-560; Scientic Industries, Bohemia, N.Y.,
U.S.A.), and CF-encapsulated liposomes were formed.
The liposomes were washed 4 times by centrifugation
(15,000 rpm, 20 min, 4 C) with PBS in order to remove
free CF and were resuspended in 2 ml of PBS.
Membrane-damaging activity. We evaluated mem-
brane-damaging activity of the seven selected com-
pounds and the two acid-treated compounds. Mem-
252 K. FUNATOGAWA ET AL
253 ANTI-H. PYLORI ACTIVITY OF HYDROLYZABLE TANNINS
Table 1. Compounds tested in this study and their plant sources
Plant source(s)
Compound M.W.
Common name (Scientic name)
Reference(s)
Hydrolyzable tannins
Monomers
Penta-O-galloyl--D-glucose 941 Chinese gall (Rhus chinensis), Eucalyptus (Eucalyptus spp.) 41
Strictinin 634 Autumn olive (Elaeagnus umbellata), Guava (Psidium guajava) 51, 53
Pedunculagin 784 Chinese agrimony (Agrimonia pilosa) 23
Tellimagrandin I (TG-I) 787 Ramanas rose (Rosa rugosa), Camellia (Camellia japonica) 1, 39, 53
Tellimagrandin II (TG-II) 939 Shan Zhu Yu (Cornus ofcinalis), Camellia (Camellia japonica) 1, 30, 53
Casuarictin 937 Drooping sheoak (Casuarina stricta) 1
Corilagin 634 Thunbergs geranium (Geranium thunbergii) 32, 38
Geraniin 953 Thunbergs geranium (Geranium thunbergii) 12, 32
Casuarinin 937 Drooping sheoak (Casuarina stricta) 36
Elaeagnatin A 1,246 Autumn olive (Elaeagnus umbellata) 18
Hippophaenin A 953 Sea buckthorn (Hippophae rhamnoides) 51
Dimers
Agrimoniin 1,871 Chinese agrimony (Agrimonia pilosa) 23
Nobotanin B 1,873 Nobotan (Melastoma candidum) 52
Rugosin D 1,875 Ramanas rose (Rosa rugosa) 39
Heterophylliin B 1,873 Siberian lbert (Corylus heterophylla) 23
Oenothein B 1,569 Evening primrose (Oenothera stricta) 26, 40
Euphorbin C 1,887 Asthma weed (Euphorbia hirta) 23
Alienanin B 1,855 Oriental white oak (Quercus aliena), Cliffrose (Cowania mexicana) 17
Trimers
Heterophylliin G 2,656 Siberian lbert (Corylus heterophylla) 23
Oenothein A 2,354 Evening primrose (Oenothera stricta) 26, 40
Catechins
()-Epicatechin (EC) 290 13, 25
()-Epicatechin gallate (ECG) 442 Green tea (Camellia sinensis) 13, 25
()-Epigallocatechin gallate (EGCG) 458 13, 25
Proanthocyanidins
Procyanidin B-1 578 19, 23
Procyanidin B-3 578 19, 23
Procyanidin B-4 578 Grape seed (Vitis vinifera), Cacao bean (Theobroma cacao), 19, 23
Procyanidin B-5 578 Loquat (Eriobotrya japonica), Apple (Malus sylvestris) 19, 23
Procyanidin C-1 866 19, 23
Procyanidin polymer 3,170 Loquat (Eriobotrya japonica) 19, 23
Other polyphenols
Chlorogenic acid 354 Cacao bean (Theobroma cacao), Loquat (Eriobotrya japonica) 24
Isorhamnetin 3-O-rutinoside 624 Blackbrush (Coleogyne ramosissima) 20
8-C-Rha-Glc-naringenin 580 Loquat (Eriobotrya japonica) 19
Eryodictyol 7-O-glucoside 450 Blackbrush (Coleogyne ramosissima) 17
Iridin 328 German iris (Iris germanica) 56
Tri-N-coumaroylspermidine 583 Blackbrush (Coleogyne ramosissima) 44
Piceatannol-4'-O-(6"-galloyl)-Glc 558 Cajeput (Melaleuca leucadendron) 50
Terpenoids
Roseoside 386 Loquat (Eriobotrya japonica) 21
3-O-Caffeoyl-betulinic acid 618 Cajeput (Melaleuca leucadendron) 6
Betulinic acid 457 Birch (Betula spp.) 2
Glycyrrhizin 822 Licorice (Glycyrrhiza spp.) 55

brane-damaging activity was determined by measuring


CF released from the liposomes (13). In a cuvett
(No./REF 67.755; Sarstedt, Numbrecht, Germany), each
test compound (3.13 to 100 g) and 25 l of liposomes
were mixed with PBS to attain a total volume of 1 ml.
The mixture was incubated at 37 C for 60 min, and u-
orescence intensity was measured at various time points
by using a uorescence spectrophotometer (650-10S;
Hitachi, Tokyo) at excitation and emission wavelengths
of 490 nm and 520 nm, respectively. The total amount of
CF in 25 l of liposomes was determined by mixing
the liposomes with 1% Triton X-100 (Wako, Osaka,
Japan) at 37 C for 30 min.
Cytotoxicity against MKN-28 cells. We assessed
cytotoxicity of three hydrolyzable tannins (TG-I, TG-II,
and Oenothein A). MKN-28 cells, derived from human
gastric carcinoma, were used for the analysis of cyto-
toxicity (28). The cells were cultured in RPMI-1640
medium (RPMI; Sigma) supplemented with 10% fetal
calf serum at 37 C under 5% CO
2
for 2 days using at-
bottom 96-well tissue culture plates (Falcon 3072; Bec-
ton Dickinson, Lincoln Park, N.J., U.S.A.). After the
cells had formed conuent monolayers, the medium
was decanted from the plates and 100 l of RPMI, alone
or containing each test compound (12.5 to 50 g/ml), was
added to each well. The plates were incubated at 37 C
under 5% CO
2
for 12 hr, and then the viability of the cells
was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide (MTT) assay (27).
Results
Antibacterial Activity
The MICs of plant-derived compounds against four H.
pylori strains and one E. coli strain are presented in
Table 2. Furthermore, the MICs of the seven selected
compounds and the two acid-treated compounds against
32 H. pylori strains are summarized in Table 3. All
hydrolyzable tannins tested demonstrated promising
antibacterial activity against H. pylori, and monomers
254 K. FUNATOGAWA ET AL
Fig. 1. Chemical structures of TG-I, TG-II, Geraniin, Agrimoniin, Oenothein A, EGCG, and EC.
255 ANTI-H. PYLORI ACTIVITY OF HYDROLYZABLE TANNINS
Table 2. Antibacterial activity of plant-derived compounds against four H. pylori strains and one E. coli strain
MIC (g/ml)
a)
Compound H. pylori E. coli
NCTC11638 ATCC43504 A-13 A-19 JC-2
Hydrolyzable tannins
Monomers
Penta-O-galloyl--D-glucose 12.5 12.5 12.5 12.5 100
Strictinin 6.25 25 6.25 6.25 100
Pedunculagin 12.5 12.5 12.5 12.5 100
Tellimagrandin I (TG-I) 12.5 12.5 12.5 12.5 100
Acid-treated TG-I 12.5 12.5 12.5 12.5 100
Tellimagrandin II (TG-II) 12.5 12.5 6.25 12.5 100
Acid-treated TG-II 12.5 12.5 12.5 12.5 100
Casuarictin 12.5 25 12.5 12.5 100
Corilagin 12.5 12.5 6.25 12.5 100
Geraniin 12.5 25 12.5 25 100
Casuarinin 25 25 12.5 25 100
Elaeagnatin A 25 25 50 50 100
Hippophaenin A 12.5 12.5 12.5 12.5 100
Dimers
Agrimoniin 25 25 25 25 100
Nobotanin B 12.5 12.5 12.5 25 100
Rugosin D 25 50 25 25 100
Heterophylliin B 12.5 25 12.5 25 100
Oenothein B 25 12.5 12.5 25 100
Euphorbin C 25 25 50 25 100
Alienanin B 25 25 25 25 100
Trimers
Heterophylliin G 12.5 25 12.5 25 100
Oenothein A 50 50 25 25 100
Catechins
()-Epicatechin (EC) 100 100 100 100 100
()-Epicatechin gallate (ECG) 50.0 100 50 100 100
()-Epigallocatechin gallate (EGCG) 25 50 25 25 100
Proanthocyanidins
Procyanidin B-1 100 100 100 100 100
Procyanidin B-3 100 100 50 100 100
Procyanidin B-4 100 100 50 100 100
Procyanidin B-5 50 100 50 25 100
Procyanidin C-1 100 100 100 100 100
Procyanidin polymer 100 100 100 100 100
Other polyphenols
Chlorogenic acid 100 100 100 100 100
Isorhamnetin 3-O-rutinoside 100 100 100 100 100
8-C-Rha-Glc-naringenin 100 100 100 100 100
Eryodictyol 7-O-glucoside 100 100 100 100 100
Iridin 100 100 100 100 100
Tri-N-coumaroylspermidine 100 100 100 100 100
Piceatannol-4'-O-(6"-galloyl)-Glc 25 50 12.5 25 100
Terpenoids
Roseoside 100 100 100 100 100
3-O-Caffeoyl-betulinic acid 25 50 50 25 100
Betulinic acid 100 100 100 100 100
Glycyrrhizin 100 100 100 100 100
a)
MICs were determined by the agar dilution method with PPLO agar supplemented with 0.2% DMCD.
revealed stronger activity than oligomers. Acid-treated
hydrolyzable tannins also showed antibacterial activity.
Acid-treatment did not signicantly affect their antibac-
terial activity (P0.05, by paired Students t test). A cat-
echin, EGCG demonstrated promising antibacterial activ-
ity against H. pylori, though other catechins did not.
Procyanidins, other polyphenols and terpenoids demon-
strated minimal antibacterial activity against H. pylori
with a few exceptions. On the other hand, no com-
pounds tested in this study revealed antibacterial activi-
ty against E. coli.
Bactericidal Activity
The killing kinetics of TG-I against H. pylori is sum-
marized in Fig. 2. TG-I demonstrated time- and dose-
dependent bactericidal activity. The number of viable H.
pylori cells decreased progressively by exposure to TG-
I and reached less than the assay limit within 24 hr.
Effect on Morphology of H. pylori
The results of morphological study are shown in Fig.
3. H. pylori cells aggregated and formed clusters by
exposure to 50 g/ml TG-I. Similar clusters were not
observed in the broth inoculated with no bacteria, which
indicates that these clusters were not aggregates of broth
components. At 2 hr after the onset of exposure, it was
possible to observe individual helical rods in the clusters
and to disperse them by pipetting. The number of viable
H. pylori cells was not changed remarkably by 2 hr
exposure to 50 g/ml TG-I (Fig. 2). Thus, most of
these helical rods must have been alive. From 8 hr
onward, only fused cells were observed in the clusters,
and it was not possible to disperse them. During this
period, the number of viable cells decreased exponen-
tially and reached less than the assay limit (Fig. 2).
Thus, these fused cells must have been dying or dead.
Membrane-Damaging Activity
TG-I damaged liposomal membranes and released
CF from the liposomes (Fig. 4). The amount of CF
released from the liposomes increased in a time-depen-
dent manner and reached a plateau after 40 min of expo-
sure to TG-I. Membrane-damaging activity of the seven
selected compounds and the two acid-treated compounds
is summarized in Fig. 5. All hydrolyzable tannins test-
ed released CF in a dose-dependent manner. Acid-treat-
ed hydrolyzable tannins also showed dose-dependent
membrane-damaging activity. Acid-treatment slightly
decreased their membrane-damaging activity (P0.01,
by paired Students t test). A catechin, EGCG also
demonstrated membrane-damaging activity that increased
from 3.13 to 12.5 g/ml in a dose-dependent manner. At
low concentrations, EGCG showed stronger membrane-
damaging activity than hydrolyzable tannins. Another
catechin, EC exhibited minimal membrane-damaging
activity.
Cytotoxicity against MKN-28 Cells
None of the hydrolyzable tannins tested demonstrated
cytotoxicity against MKN-28 cells (Table 4).
Discussion
In this study, we investigated antibacterial activity of
40 plant-derived compounds against H. pylori in vitro.
All hydrolyzable tannins tested demonstrated promising
antibacterial activity against H. pylori. Monomeric
hydrolyzable tannins revealed especially strong activity.
256 K. FUNATOGAWA ET AL
Table 3. Antibacterial activity of seven selected compounds and
two acid-treated compounds against 32 H. pylori strains
Compound
No. of
MIC (g/ml)
a)
strains
Range MIC50
b)
MIC90
c)
TG-I 32 3.1312.5 12.5 12.5
Acid-treated TG-I 32 3.1312.5 12.5 12.5
TG-II 32 3.1312.5 12.5 12.5
Acid-treated TG-II 32 6.2512.5 12.5 12.5
Geraniin 32 6.2525 25 25
Agrimoniin 32 12.550 25 50
Oenothein A 32 12.550 50 50
EGCG 32 12.550 25 50
EC 32 1001,600 400 800
a)
MICs were determined by the agar dilution method with
PPLO agar supplemented with 0.2% DMCD.
b)
MIC at which 50% of the strains are inhibited.
c)
MIC at which 90% of the strains are inhibited.
Fig. 2. Bactericidal activity of TG-I against H. pylori. H. pylori
NCTC 11638 was exposed to TG-I at concentrations of 50 (), 25
(), 12.5 (), and 0 () g/ml. Samples were taken at the
times indicated, and viability was measured by the plate colony
count technique.
257 ANTI-H. PYLORI ACTIVITY OF HYDROLYZABLE TANNINS
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A catechin, EGCG also demonstrated antibacterial activ-
ity. However, other compounds exhibited minimal
antibacterial activity against H. pylori with a few excep-
tions. Hydrolyzable tannins have higher water-solubil-
ity than other compounds (53). Water-solubility may be
important for their antibacterial action. Mabe et al.
have reported that tea catechins may have therapeutic
effects on H. pylori infection (25). In this study, many
hydrolyzable tannins revealed stronger antibacterial
activity than catechins. Furthermore, acid-treatment
scarcely affected the antibacterial activity of hydrolyzable
tannins in vitro, which suggests that they can work in the
acidic gastric environment. Thus, hydrolyzable tannins
may be promising therapeutic agents in the treatment of
H. pylori infection.
No compounds tested in this study demonstrated
antibacterial activity against E. coli which is a normal
inhabitant of the human intestinal tract. Hydrolyzable
tannins may have a narrow antibacterial spectrum. New
triple therapies suppress not only H. pylori but also
intestinal bacterial ora, which causes such side effects
as abdominal pain and diarrhea (46). Harsch et al. have
reported a case of pseudomembranous colitis after erad-
ication of H. pylori with a new triple therapy (9).
Hydrolyzable tannins may be able to suppress H. pylori
without affecting intestinal bacterial ora. Furthermore,
hydrolyzable tannins did not affect the viability of MKN-
28 cells derived from human gastric epithelium (28).
Thus, hydrolyzable tannins may be able to suppress H.
pylori without affecting the viability of gastric epithelial
cells.
A monomeric hydrolyzable tannin, TG-I decreased the
viability of H. pylori in time- and dose-dependent man-
ners in vitro. This result indicates that hydrolyzable
tannins have bactericidal effects against H. pylori. In the
process of bacterial killing, TG-I rapidly aggregated H.
pylori cells into clusters and fused them, which sug-
gests that hydrolyzable tannins act on the surface struc-
tures of H. pylori. Ikigai et al. have reported antibacte-
rial catechins damage bacterial membranes (13). Then,
258 K. FUNATOGAWA ET AL
Fig. 4. Membrane-damaging activity of TG-I. CF-encapsulated
liposomes were exposed to TG-I (50 g/ml). Samples were
taken at the times indicated, and the amount of CF released from
the liposomes was measured. Data are presented as means
standard deviations.
Fig. 5. Membrane-damaging activity of TG-I (), TG-II (),
acid-treated TG-I ( ), acid-treated TG-II ( ), Geraniin (),
Agrimoniin (), Oenothein A (), EGCG (), and EC (). CF-
encapsulated liposomes were exposed to each compound (3.13 to
100 g/ml). Samples were taken after 40 min of exposure, and
the amount of CF released from the liposomes was measured.
Data are presented as means standard deviations.
Table 4. Effect of plant-derived compounds on the viability of
MKN-28 cells
a)
Compound Concentration Viability (OD540)
b)
RPMI alone 0.930.06
12.5 g/ml 0.890.08
TG-I 25 g/ml 0.930.06
50 g/ml 0.940.05
12.5 g/ml 0.870.09
TG-II 25 g/ml 0.870.08
50 g/ml 0.850.09
12.5 g/ml 0.890.04
Oenothein A 25 g/ml 0.870.08
50 g/ml 0.870.08
a)
MKN-28 cells were treated with RPMI alone or containing
each compound (12.550 g/ml) for 12 hr.
b)
The viability of MKN-28 cells is expressed as the OD at 540
nm (OD540). Each value represents the mean the standard
deviation. There was no signicant difference between each
compound and RPMI alone (P0.05 by unpaired Students t test).
we investigated effects of hydrolyzable tannins and cat-
echins on lipid bilayer membranes. All hydrolyzable tan-
nins tested demonstrated dose-dependent membrane-
damaging activity. An antibacterial catechin, EGCG
showed stronger membrane-damaging activity than
hydrolyzable tannins, though it was inferior to many
hydrolyzable tannins in antibacterial activity. On the
other hand, hydrolyzable tannins did not reveal toxicity
against E. coli and MKN-28 cells.
H. pylori membrane contains three kinds of cholesteryl
glucosides (CGs); cholesteryl--D-glucopyranoside
(CGL), cholesteryl-6-O-tetradecanoyl--D-glucopyran-
oside (CAG), and cholesteryl-6-O-phosphatidyl--D-
glucopyranoside (CPG) (7, 11). These glycolipids
account for about 25% (wt/wt) of the total lipid. CGs are
found in many plants, but they are very rare in bacteria
and animals. Especially, CPG is a unique lipid found
only in Helicobacter species (8). These unique mem-
brane features might be related to the sensitivity of H.
pylori to hydrolyzable tannins.
In conclusion, plant-derived hydrolyzable tannins
have antibacterial effects against H. pylori. Hydrolyzable
tannins have potential as new and safe therapeutic regi-
mens against H. pylori infection. Whether their mem-
brane-damaging activity directly contributes to their
antibacterial action remains to be elucidated.
This study was supported in part by a Grant-in-Aid for Sci-
entic Research (No. 09672143) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
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261 ANTI-H. PYLORI ACTIVITY OF HYDROLYZABLE TANNINS

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