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Antimicrobial Agents and Chemotherapy, July 1999, p. 1788-1791, Vol. 43, No. 7
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro and In Vivo Activities of Tea Catechins
against Helicobacter pylori
Katsuhiro
Mabe,1,*
Masami
Yamada,2
Itaro
Oguni,3 and
Tsuneo
Takahashi1
Second Department of Internal Medicine,
Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata
City, Yamagata 990-9585,1
Gastrointestinal Division, Hamamatsu Medical Center, Hamamatsu
City, Shizuoka 432-8002,2 and
Department of Food and Nutritional Sciences, University of
Shizuoka Hamamatsu College, 3-2-3 Nunohashi, Hamamatsu City,
Shizuoka 432-8012,3 Japan
Received 21 September 1998/Returned for modification 13 January
1999/Accepted 19 April 1999
 |
ABSTRACT |
The catechin epigallocatechin gallate showed the strongest activity
of the six tea catechins tested against Helicobacter pylori (MIC for 50% of the strains tested, 8 µg/ml). It had bactericidal activity at pH 7 but not at pH
5.0. In infected Mongolian gerbils, H. pylori was eradicated in 10 to 36% of the
catechin-treated animals, with significant decreases in mucosal
hemorrhage and erosion. Tea catechins, therefore, may have therapeutic
effects on H. pylori infection.
 |
TEXT |
The association between
Helicobacter pylori infection and upper gastrointestinal
diseases such as chronic gastritis, peptic ulceration, and gastric
cancer has been widely investigated (13, 22, 25). H. pylori is sensitive to various antibiotics in vitro (5,
11). However, clinical trials with such an antibacterial agent
alone have mostly failed to eradicate H. pylori (3,
17). Although the new triple therapy consisting of the combined
use of two antibiotics and a proton pump inhibitor suppressing acid secretion shows a high eradication rate and a low incidence of harmful
side effects (2, 19), some problems remain. In recent years,
for instance, an increased occurrence of metronidazole- and/or
clarithromycin-resistant strains of H. pylori has become a
problem (12, 15, 18). This problem might be amplified in
Asia and Africa, where many people are infected with H. pylori (1, 6, 16). Even if the widespread use of
antibiotics were feasible economically and logistically in these
countries, it would most certainly lead to increased resistance of not
only H. pylori but also other pathogenic bacteria.
Therefore, a nonantibiotic agent which is both highly effective and
safe might be of utmost importance for the eradication of both
antibiotic-susceptible and -resistant strains of H. pylori.
Recent studies have presented data that show a variety of biological
activities of tea catechins, compounds which constitute about 15% (dry
weight) of green tea (7). It has been reported that tea
catechins have antibacterial activity against various foodborne
pathogenic bacteria (8). Thus, it seems reasonable to
explore the possibility of using tea catechins, harmless compounds extracted from green tea, for eradication of H. pylori. In
this study, we investigated the antibacterial activity of catechins against H. pylori in vitro and in vivo and the in vivo
effect of these compounds on the gastric mucosal injury induced by this organism in Mongolian gerbils.
Bacterial strains.
Two standard strains (ATCC 43504 and ATCC
43629) and 108 clinical isolates (YMA1 to YMA108) of H. pylori were used. The clinical isolates were obtained from gastric
biopsy specimens from patients with gastritis and peptic ulcer, and
their identification was based on standard biochemical tests
(14). Stock cultures were stored at
80°C in
Mueller-Hinton broth (Difco Laboratories, Detroit, Mich.) supplemented
with 10% heat-inactivated horse serum (Nacalai Tesque, Kyoto, Japan).
Catechins.
Epigallocatechin gallate (EGCg), epicatechin
gallate, epigallocatechin, epicatechin, crude catechin (Polyphenon
70SR), and crude theaflavin were from Mitsui Norin
(Shizuoka, Japan). Each was more than 98% pure, excluding the crude
compounds. The structural formulas of tea catechins are shown in Fig.
1. Polyphenon 70SR contains a
total of 73.4% catechins, including EGCg (33.5%), epicatechin gallate
(10.1%), epigallocatechin (17.4%), and epicatechin (8.6%).
Theaflavins are made up of the dimers of catechins which are produced
during the manufacture of black tea.
In vitro studies.
MICs were determined by a broth
microdilution method with Mueller-Hinton broth supplemented with 10%
heat-inactivated horse serum and an inoculum of 5 × 104 CFU per well. Broth microdilution plates were prepared
in house and stored at
80°C until use. After bacterial inoculation,
plates were incubated for 72 h and then MICs were determined.
Bactericidal actions were determined by using an in vitro killing
assay. A bacterial suspension of an H. pylori clinical
isolate, YMA78 (100 µl, 107 CFU) was inoculated into 10 ml of culture medium containing EGCg concentrations equal to 0, 0.25, 1, 2, and 4 times the MIC (32 µg/ml) and incubated for 48 h at
37°C in an atmosphere of 5% O2-10% CO2-85% N2 with reciprocation. Samples (100 µl) were taken 0, 3, 6, 12, 24, and 48 h after the start of
incubation for viable-cell counting. Viability was measured by the
plate colony count technique. Colonies were counted after 5 days of
incubation. Effects of pH on the antibacterial activity of EGCg were
assessed with time-kill curves at pH values of 7.0 (67 mM Sorensen
phosphate buffer), 5.0, and 4.0 (30 mM citrate buffer) by using the
method reported previously (23). The final concentrations of
EGCg were 0, 250, and 500 µg/ml. The cultures were incubated at
37°C for 80 min with reciprocation. Samples (100 µl) were taken 0, 20, 40, and 80 min after the start of incubation for viable-cell counting.
Animals and inoculation with H. pylori.
Seven-week-old,
specific-pathogen-free, male Mongolian gerbils (MGS/sea; body weight,
40 to 50 g) purchased from Seac Yoshitomi (Fukuoka, Japan) were
used in this study. The gerbils were housed in animal facilities, fed a
sterilized commercial rodent diet (CE-2; Japan CLEA, Tokyo, Japan), and
allowed free access to sterilized distilled water. Each animal was
fasted for 24 h and inoculated orally with a suspension of
H. pylori ATCC 43504 (500 µl, 108 CFU) by
using a feeding needle (9). After inoculation, each animal
was kept without food and water for 4 h.
Evaluation of catechins in vivo. (i) Experiment A.
Gerbils
were randomly divided into four groups of 9 or 10 after bacterial
inoculation. Four weeks after bacterial inoculation, each group of
gerbils was fed a different diet (i.e., containing 0, 0.5, 1, or 2%
catechins) for 2 weeks. These diets and water were provided ad libitum
throughout the test period. After the animals were fasted for 24 h, their stomachs were excised and cut along the greater curvature for
macroscopic observation. The stomachs were then homogenized in 10 ml of
saline. A 100-µl aliquot each of serial dilutions of the homogenate
was spread on an M-BHM PYLORI AGAR plate (Nikken Bio Medical
Laboratory, Kyoto, Japan). The plates were incubated for 5 days, and
the colonies were counted.
(ii) Experiment B.
Gerbils were randomly divided into three
groups of 10 or 11 after bacterial inoculation. Six weeks after
inoculation, the gerbils were treated for 2 weeks as follows: (i)
control without catechins, (ii) 1% catechin-containing diet, and (iii)
1% catechin-containing diet plus 0.5% catechin-containing water.
After the gerbils were fasted for 24 h, their stomachs were
excised and homogenized. Colonies were counted as described above.
Statistical analysis.
A one-way analysis of variance (ANOVA),
Scheffe's test, and the Kruskal-Wallis test were used. The level of
significance selected was P < 0.05.
The MICs of six catechins against H. pylori isolates are
presented in Table 1. All of the
catechins showed activity against H. pylori, and EGCg showed
the strongest activity of all of the catechins tested. The bactericidal
action of EGCg was also examined. Figure
2 shows representative data obtained with
strain YMA78 exposed to 8, 32 (MIC), 64, and 128 µg of EGCg per ml.
Bacteriostatic and bactericidal effects were observed at concentrations
equal to 2 and 4 times the MIC, respectively. Although the mechanism of
this action is still obscure, structure-activity relationship studies
indicated that the antibacterial activities of catechins were
predominantly related to the gallic acid moiety and the number of
hydroxyl groups (10; Fig. 1). It has also been
reported that catechins damage the membrane lipid bilayer
(10). Catechins probably damage the membrane of H. pylori, but further aspects of their effect, such as morphological
changes, should be tested. Moreover, EGCg inhibits the urease activity
and motility of H. pylori (data not shown), which may
contribute to its antibacterial activity in vivo. As shown in Fig.
3, H. pylori YMA78 survived for 80 min in all of the buffers used at pHs 4.0 to 7.0. EGCg showed a
dose-dependent bactericidal action at pH 7.0 but only a weak effect at
pHs 4.0 and 5.0, indicating that the bactericidal action of EGCg
against H. pylori was pH dependent. The activity of
antibiotics such as clarithromycin decreases under acidic conditions (4). These data suggest that the bactericidal action of EGCg is weakened by acidic conditions.

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FIG. 2.
Effect of EGCg on H. pylori viability in
liquid medium. H. pylori YMA78 was cultured microaerobically
in Mueller-Hinton broth supplemented with 10% heat-inactivated horse
serum at 37°C with reciprocation in the presence of EGCg at
concentrations of 128 ( ), 64 ( ), 32 ( ), 8 ( ), and 0 ( )
µg/ml. Culture samples (100 µl) were taken at the times indicated,
and viability was measured by the plate colony count technique.
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FIG. 3.
Effect of EGCg on H. pylori viability in
buffers at various pHs. H. pylori YMA78 was incubated
microaerobically in each buffer at 37°C with reciprocation in the
presence of EGCg at concentrations of 500 ( ), 250 ( ), and 0 ( )
µg/ml. Viability was determined at each time point indicated.
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The effect of catechin on H. pylori colonization in vivo is
shown in Table 2. In experiment A,
H. pylori was eradicated in about 10% of the gerbils in
each of the catechin-fed groups whereas H. pylori was
detected in all of the control gerbils. When a one-way ANOVA was
performed to compare the number of H. pylori cells in the
stomachs of the gerbils (excluding those in which the bacteria had been
eradicated), significant differences among the four groups were found.
However, on comparison of each group with the control by using
Scheffe's test, only the 0.5% catechin-fed group showed a significant
decrease. In experiment B, H. pylori was eradicated in 10%
of the gerbils in the 1% catechin-containing diet group (catechin at
70 mg/head/day) and 36.4% of the gerbils given the 1%
catechin-containing diet plus 0.5% catechin-containing water (catechin
at 100 mg/head/day) while H. pylori was detected in all of
the control animals. Excluding the gerbils in which the bacteria had
been eradicated, a one-way ANOVA was used to compare the numbers of
H. pylori cells in the stomachs and no significant differences among the three groups were found. In our infection model,
tea catechins showed an antibacterial effect but only at a low
eradication rate of 10 to 36.4%. Similarly, eradication therapies
using single antibiotics also result in failure in humans despite their
in vitro efficacy (3, 17). The pH dependency of
antibacterial activity may be one of the factors producing such low
eradication rates. Combinations of catechins with a proton pump
inhibitor which neutralizes the acidity in the stomach might be
effective, as in the new triple therapy.
Another reason why catechins are not significantly effective in the
eradication of H. pylori might be the short gastric-transit time of these agents. The effect on eradication was enhanced when the
catechins were administrated in both the diet and drinking water, while
no additional effect was obtained with a higher-dose catechin diet.
Thus, further studies on efficacy may be warranted in which catechins
are combined with a proton pump inhibitor and a drug delivery system
which will prolong the gastric-transit time is used. Macroscopic
findings on the gastric mucosa of the H. pylori-infected
gerbils in the first in vivo experiment are shown in Table
3. At 6 weeks after H. pylori
infection, notable changes in the antral and fundic mucosa, such as
hemorrhages and erosions, were observed in all control animals. The
hemorrhage scores and the scores of injury to the gastric mucosa were
significantly decreased (P < 0.01 versus the control)
in all of the catechin-fed groups, although the bacteria were not
eradicated in most of the animals tested. This decrease may have
derived primarily from the antibacterial and antiurease effects of tea
catechins, although it is possible that other actions for which
catechins are known, such as antioxidative (21) or
anti-inflammatory (24) effects or inhibition of gastric acid
secretion (20), also contribute, in part, to this efficacy.
In conclusion, tea catechins have an antibacterial effect against
H. pylori and may have a therapeutic effect against gastric mucosal injury induced by this organism. A new, safe, and effective therapeutic regimen against H. pylori infection may be
contrived by the use of catechins combined with a proton pump
inhibitor, possibly in a delivery system which prolongs the
gastric-transit time of catechins.
 |
ACKNOWLEDGMENTS |
We thank Y. Hara (Food Research Institute, Mitsui Norin Co., Ltd.)
for kindly providing catechins and T. Ishigami (Food Research Institute, Mitsui Norin Co., Ltd.) for his skillful technical collaboration.
This study was partly supported by a grant from the Program for
Promotion of Basic Research Activities for Innovative Biosciences in Japan.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Second
Department of Internal Medicine, Yamagata University School of
Medicine, 2-2-2 Iida-Nishi, Yamagata City, Yamagata 990-9585, Japan.
Phone: 81-23-628-5309. Fax: 81-23-628-5311. E-mail:
kmabe{at}med.id.yamagata-u.ac.jp.
 |
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Antimicrobial Agents and Chemotherapy, July 1999, p. 1788-1791, Vol. 43, No. 7
0066-4804/99/$04.00+0
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