Aliment Pharmacol Ther 2000; 14: 635±638.
Anti-Helicobacter pylori activity of Chinese tea: in vitro study
YUK -KEI Y EE* & MARCEL WING -LEUNG KOO 
*Department of Medicine, Tuen Mun Hospital, Hong Kong and  Department of Pharmacology, Faculty of Medicine,
University of Hong Kong, Hong Kong
Accepted for publication 23 December 1999
SUMMARY
Background: Chinese tea has an antibacterial activity
against a wide range of bacteria. However, its activity
against Helicobacter pylori has not been reported.
Method: In this study the anti-Helicobacter pylori
effects of a Chinese tea (Lung Chen tea), and two
tea catechins, epigallocatechin gallate and epicatechin
and their minimum inhibitory concentrations (MICs)
were examined. The effect of Lung Chen on
metronidazole resistance was also studied using the
E-test.
INTRODUCTION
Helicobacter pylori, which was ®rst discovered in 1982,
was found to be an important factor in the pathogenesis
of peptic ulcer disease, gastritis, and gastric malignancy.1
The Maastricht Consensus Report has recommended
H. pylori eradication in many diseases and conditions.2
Proton-pump inhibitor based triple therapy is now the
most commonly accepted eradication regimen.3 How-
ever, antibiotic resistance is not uncommon; metroni-
dazole resistance is a well-known issue and can be as
high as 60±70% in some areas.4, 5 Clarithromycin
resistance is also an increasing problem as its use has
become more popular in recent years.5
Tea is a widely consumed beverage, especially by the
Chinese and Japanese. Many studies have shown its
anticarcinogenic, antimitagenic, antioxidative and hypo-
cholesterolaemic effects.6±10 The use of tea in soldiers'
Correspondence to: Dr Yuk-kei Yee, Department of Medicine, Tuen Mun
Hospital, Tuen Mun, New Territory, Hong Kong.
E-mail: ykyee@bigfoot.com
Ó 2000 Blackwell Science Ltd
Results: Lung Chen, epigallocatechin gallate and epi-
catechin all inhibited the growth of H. pylori. The MIC90
for Lung Chen was 0.25±0.5% (w/w) and that of
epigallocatechin gallate and epicatechin were 50±100
and 800±1600 lg/mL, respectively. Epigallocatechin
gallate is probably the active ingredient responsible for
most of the anti-H. pylori activity of Chinese tea. Lung
Chen did not reverse metronidazole resistance.
Conclusions: Chinese tea has anti-H. pylori activity in a
daily consumed concentration, and epigallocatechin
gallate is probably the active ingredient responsible for
the action.
water bottles as a prophylaxis for typhoid was reported
many decades ago.11 Recently studies found that tea
extracts and catechins inhibited and killed a wide range
of bacteria.12±15 However, no report of their effects on
H. pylori is available. This study investigated whether
the Chinese tea, Lung Chen, had any anti-H. pylori
effect. The minimum inhibitory concentrations of Lung
Chen and two tea catechins, epigallocatechin gallate
and epicatechin were also studied. Epigallocatechin
gallate and epicatechin were tested because previous
studies have demonstrated the greatest and least
(respectively) pharmacological and antibacterial
activities.16 The synergistic effect of Lung Chen on
metronidazole resistance was also examined.
METHODS
Helicobacter pylori isolates
Twenty clinical isolates of H. pylori were isolated from
patients attending Tuen Mun Hospital with dyspepsia
and/or peptic ulcer in March and April 1998. Stock
635
636 YUK-KEI YEE & M. WING-LEUNG KOO
cultures were maintained on a protected bead system
and deep-frozen at )80 °C on horse blood broth. Before
the studies, H. pylori were prepared by sub-culturing
them onto Mueller±Hinton Agar supplemented with 5%
de®brinated horse blood and incubated for 48 h micro-
aerobically.
Tea and catechin solutions
Tea solutions were prepared by soaking the tea leaves in
0.05 M phosphate-buffered saline for 30 min.10 The
supernatant, after adjustment to pH 7.0 with 2.5 mol/L
NaOH, was used as the tea extracts.12, 13 The Lung
Chen tea was obtained from West Lake of Hangzhou,
China. Epigallocatechin gallate (Sigma) and epicatechin
(Sigma) solutions were prepared by dissolving the pure
compounds in phosphate-buffered saline.
Screening of susceptibility of H. pylori to Lung Chen tea
The screening was performed with seven H. pylori clinical
isolates. Three wells (7 mm diameter) were punched in
each agar plate using a stainless steel borer; two plates
were used for each isolate. Each well was ®lled with
0.1 mL of 1%, 2%, 4%, 8% or 16% Lung Chen tea extract,
while the last well was ®lled with phosphate-buffered
saline as control. The plates were incubated at 37 °C
under microaerophilic conditions for 72 h (85% N2, 10%
CO2, 6% O2; Oxoid gas-generating kit and GasPark jars
with CampyPak Plus). The presence or absence, and the
size of the inhibitory zone were measured.
Measurement of minimum inhibitory concentrations
Minimum inhibitory concentration was performed with
the plate dilution method according to recommenda-
tions of the National Committee for Clinical Laboratory
Standards, modi®ed for testing H. pylori. Double dilu-
tions of epigallocatechin gallate, epicatechin or Lung
Chen in 1 mL aliquots were added to 24 mL of melted
Mueller±Hinton Agar supplemented with 5% de®bri-
nated horse blood to prepare a set of agar plates
containing double dilutions of these materials. Twenty
isolates of H. pylori were used. The bacteria were
suspended in sterile nutrient broth to obtain a turbidity
equivalent to a 0.5 McFarland standard (108/mL).
Bacteria were inoculated onto the plates with a
multipoint inoculator (Denley, Liverpool, UK) which
delivered an inoculum of approximately 104±105 CFU
per spot as recommended by previous investigators. The
results were evaluated after 72 h of incubation. Mini-
mum inhibitory concentration was quoted as the lowest
concentration at which no growth was seen. The
overall MIC50 and MIC90 (levels in which the growth
of 50% and 90% or more strains were inhibited) for the
20 isolates of H. pylori were then determined.
Synergistic effects of tea extracts on the minimum
inhibitory concentration of metronidazole
The assay was performed with the E-test, which was
found to be acceptable for metronidazole, although not
as good as for ampicillin and clarithromycin.17 The test
was performed using two types of agar plates (the
standard Mueller±Hinton Agar and the test plate) and
10 clinical isolates of H. pylori. Besides containing
0.0625% tea extract, the test plate was the same as the
standard plate. This concentration was chosen because
it was half of the MIC90 value as obtained in this study.
After inoculating the H. pylori onto the plate and drying
for 3±4 min, the plastic E-test strip containing a
continuous exponential gradient of metronidazole were
applied to the agar plate. The results were evaluated
72 h later. Minimum inhibitory concentrations were
determined by noting the intercept of the zone of
inhibition with the graded values on the E-test strip.
RESULTS
The inhibition zone for each isolate and tea concentra-
tion was shown in Table 1. As evidenced by the size of
the inhibition zone, there was more de®nite inhibition
with increasing concentration of the Lung Chen.
Phosphate-buffered saline, as a negative control, did
not inhibit the growth of H. pylori; therefore, Lung Chen
does exert anti-H. pylori activity.
For the determination of minimum inhibitory concen-
trations of epigallocatechin gallate and epicatechin,
results were available in only 19 isolates because 1 of
the 20 isolates did not grow. The cumulative frequency
of inhibition was shown in Figure 1. The MIC50 and
MIC90 for epigallocatechin gallate were in the range
12.5±25 lg/mL and 50±100 lg/mL, respectively, while
those of epicatechin were in the range 400±800 lg/mL
and 800±1600 lg/mL, respectively. This means that
epigallocatechin gallate is about 16 times more potent
than epicatechin in terms of anti-H. pylori activity. As
epigallocatechin gallate is a larger molecule than
Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 635±638
 ANTI-H. PYLORI ACTIVITY OF CHINESE TEA 637

Table 1. Effects of different concentrations of Lung Chen tea Table 3. Comparison between MIC90 of catechins and Lung
on the diameter of the inhibition zone of Helicobacter pylori Chen tea

Concentration of Lung Chen tea Catechins MIC90

H. pylori isolates 1% 2% 4% 8% 16% Epigallocatechin gallate in Lung 83.5 lg/mL±167 lg/mL

Chen tea
H. pylori 1 A A 14 mm 20 mm 24 mm
Epicatechin in Lung Chen tea 17.8 lg/mL±35.5 lg/mL
H. pylori 2 A 14 mm 22 mm 27 mm 32 mm
Epigallocatechin gallate 50 lg/mL±100 lg/mL
H. pylori 3 A 16 mm 24 mm 28 mm 34 mm
Epicatechin 800 lg/mL±1600 lg/mL
H. pylori 4 A 18 mm 22 mm 26 mm 34 mm
H. pylori 5 24 mm 28 mm 28 mm 32 mm 36 mm
H. pylori 6 A 10 mm 16 mm 20 mm 26 mm
H. pylori 7 A A 18 mm 20 mm 22 mm chin gallate and epicatechin contents in these concen-
trations of Lung Chen, i.e. 0.125%, 0.25% and 0.5%
A = absence of inhibitory zone.
can be calculated and are presented in Table 3.11 It
was found that the epigallocatechin gallate contents in
Lung Chen at MIC90 concentrations correlated well
with the respective value in pure epigallocatechin
gallate. However, this was not the case for epicatechin;
epigallocatechin gallate is likely to be responsible for the
anti-H. pylori activity of Lung Chen.
Out of nine viable isolates, eight showed metronidazole
resistance. This re¯ects the high rate of metronidazole
resistance in Hong Kong. The addition of Lung Chen in
a concentration of 0.0625% failed to reverse the
metronidazole resistance and did not affect the mini-
Figure 1. Cumulative frequency of Helicobacter pylori growth mum inhibitory concentration of the sensitive strain.
suppression by epigallocatechin gallate (m) and epicatechin (d).

epicatechin, its relative potency will be even greater

when compared in molar ratio.
During the determination of minimum inhibitory
concentration of Lung Chen, three isolates were dead.
The growth of H. pylori was inhibited in all cases by a
concentration of 0.5%; the cumulative frequency of
inhibition is shown in Table 2. The MIC50 and MIC90
for Lung Chen were between 0.125% to 0.25% and
0.25% to 0.5%, respectively. These are much lower
than the usual cup-of-tea concentration of 2±4%.
As the catechin contents of Lung Chen have been
determined previously by Yang et al., the epigallocate-
Table 2. Cumulative frequency of Helicobacter pylori growth suppression by Lung Chen tea in 17 viable isolates and the
corresponding epigallocatechin gallate and epicatechin contents
DISCUSSION
Catechins, which constitute 5±15% of the dry weight of
Chinese tea, are simple well-characterized iso¯ava-
noids.10 They include mainly epicatechin, epicatechin
gallate, epigallocatechin and epigallocatechin gallate.
They are readily extracted from tea by hot water
infusion and account for many bene®cial effects of tea.18
Gallocatechins are characterized by the presence of
three hydroxyl groups on the `b' ring, and catechin
gallates are gallic acid esters of the hydroxyl group on
the pyran ring.19 Epicatechin gallate and epigallocate-

Concentration of Lung Chen

0.0625% 0.125% 0.25% 0.5%

Epigallocatechin gallate content 20.9 lg/mL 41.8 lg/mL 83.5 lg/mL 167 lg/mL
Epicatechin gallate content 4.4 lg/mL 8.9 lg/mL 17.8 lg/mL 35.5 lg/mL
Frequency of inhibition 0% (0) 35% (6) 71% (12) 100% (17)

Numbers in brackets are the number of H. pylori isolates that have been suppressed. The epigallocatechin gallate and epicatechin contents in
Lung Chen tea have been determined to be 3.34% and 0.71%, respectively.11

Ó 2000 Blackwell Science Ltd, Aliment Pharmacol Ther 14, 635±638

638 YUK-KEI YEE & M. WING-LEUNG KOO
chin gallate are especially important among various
catechins.16 Ikigai et al. investigated the mechanism of
bactericidal properties of catechins.20 They showed that
bactericidal catechins primarily acted on and damaged
bacterial membranes. Gram-positive bacteria such as
S. aureus were more susceptible to catechins than gram-
negative bacteria such as E. coli, and epigallocatechin
gallate was much stronger than epicatechin in bacte-
ricidal activity.20 Whether H. pylori is affected in this
manner has not been examined.
This study showed that the Chinese tea, Lung Chen, at
concentrations of 0.5% suppressed the growth of
H. pylori. As noted previously, epigallocatechin gallate
is probably the active ingredient accounting for the
anti-H. pylori effect of Lung Chen. Although Lung Chen
is effective against H. pylori in a daily consumed
concentration, we cannot yet conclude that it can
eradicate H. pylori under in vivo conditions. H. pylori
resides inside the mucus of the stomach and this may
protect the bacteria from the effect of the tea.
The Chinese and Japanese do have a high rate of
gastric cancer and ulcer despite their traditional habits
of tea drinking. Tea and catechins are de®nitely not a
complete answer for H. pylori, since other factors such
as hygiene and nutrition are important. This study also
con®rmed the high rate of metronidazole resistance in
Hong Kong, and Lung Chen in a concentration of
0.0625% has no synergistic effect with metronidazole;
further study may have to be designed to evaluate its
synergistic effect when used in higher concentrations.
As tea has been heavily consumed for thousand of years
and no major adverse effect has been noticed, tea and
catechins probably have a high therapeutic index. The
roles of Chinese tea and catechins in the prevention and
treatment of H. pylori infection are worth further
exploration.
ACKNOWLEDGEMENTS
This study had been presented in the 2nd International
Workshop on Helicobacter pylori, 24±25 April 1999,
Hong Kong.
I would like to thank Dr TL Que and Eric for their kind
assistance in the study.
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