Ji-Eun Shin

Jong-Mi Kim
Eun-Ah Bae
Yang-Jin Hyun
In Vitro Inhibitory Effect of Flavonoids on Growth,
Dong-Hyun Kim Infection and Vacuolation of Helicobacter pylori

Original Paper
Abstract prove gastric cell death by inhibiting apoptotic signaling by HP
VacA toxin.
Flavonoids, which are main constituents of herbal medicines,
have been reported to inhibit the growth of Helicobacter pylori Key words

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(HP). Therefore, to evaluate the anti-HP activity of some flavo- Helicobacter pylori ´ VacA toxin ´ procaspase-3 ´ flavonols ´ fla-
noids (flavanols, flavones, flavonols and isoflavonoids), their ef- vones ´ flavanols ´ isoflavonoids
fects on the growth and vacuolation of HP as well as the infective
properties of HP against HeLa cells were investigated. Catechins,
quercetin and naringenin weakly inhibited the growth of HP, but Abbreviations
all tested compounds did not inhibit HP infection into KATO III HP: Helicobacter pylori
cells and HP urease activity. Quercetin and naringenin inhibited BSA: bovine serum albumin
HP VacA vacuolation in HeLa cells with IC50 values of 0.046 and ESL: enhanced chemiluminescence
0.36 mM, respectively. Quercetin also inhibited procaspase-3 ac- MIC: minimum inhibitory concentration
tivation to caspase-3 in HeLa cells induced by HP VacA toxin, MTT: methylthiazolyldiphenyl-tetrazolium bromide
which may induce cell death via the proteolytic activation of a PBS: phosphate-buffered saline
cascade of caspases. However, quercetin did not affect Bax and VacA: Vacuolating cytotoxin 197
Bcl-2 protein levels. Based on these findings, quercetin may im-

Introduction
Helicobacter pylori (HP) was isolated from the gastric antrum of
chronic gastritis patients by Warren and Marshall in 1983 [1].
Pathogenic HP produces urease and vacuolating toxin strongly.
HP urease hydrolyzes urea to CO2 and ammonia. This ammonia
generated by HP protects itself from the environment of gastric
acid in the stomach, and damages directly the gastric mucosal
cell [2]. HP vacuolating toxin (VacA toxin) may be potentiated
by urease-mediated ammonia production [2], [3], [4]. Jones et
al. [5] demonstrated that HP induced epithelial gastric cell apop-
tosis. Boquet et al. reported that the apoptosis originated from its
VacA toxin and was related to caspase-3 activation [6]. Maeda et
al. reported that Bcl-2 and Bax, which are associated with mito-
chondria-related apoptosis, was not affected by VacA toxin of HP
[7]. Therefore, the inhibitions of HP growth, infection, urease,
and VacA toxin vacuolation are important for the treatment of
patients with gastritis and peptic ulcer.
There have been various studies on the useful effects of natural
medicinal components, such as flavonoids and polyphenols,
against HP growth [8], [9], [10] as well as anti-inflammatory activ-
ity [11], [12]. However, the inhibitory effects of flavonoids on infec-
tion and vacuolation of HP have not been thoroughly studied.

Affiliation
College of Pharmacy, Kyung Hee University, Seoul, Korea

Correspondence
Prof. Dong-Hyun Kim ´ College of Pharmacy ´ Kyung Hee University ´ 1 Hoegi ´ Dongdaemun-ku ´
Seoul 130-701 ´ Korea ´ Phone: +82-2-961-0374 ´ Fax: +82-2-966-3885 ´ E-mail: dhkim@khu.ac.kr

Received June 22, 2004 ´ Accepted October 14, 2004

Bibliography
Planta Med 2005; 71: 197±201 ´  Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2005-837816
ISSN 0032-0943
 Therefore, we have examined the in vitro inhibitory effects of
some flavonoids on the growth and vacuolation of HP as well as
the infective properties of HP against HeLa cells.
Materials and Methods
Materials
Bacto agar and brucella broth were purchased from Difco Labora-
tories (USA). Fetal bovine serum (FBS), horse serum and antibio-
tic-antimycological solution were obtained from Gibco BRL
(USA). Epicatechin (> 98 %), epigallocatechin (> 98 %), epicatechin
gallate (> 98 %), epigallocatechin gallate (> 80 %), naringin
(> 95 %), naringenin (> 95 %), daidzin (> 95 %), daidizein (> 98 %),
Original Paper
heparin, acetohydroxamic acid, cell culture media, neutral red,
and bovine serum albumin (BSA) were acquired from Sigma Che-
mical Co. (USA). Baicalin (> 98 %) and baicalein (> 98 %) were pur-
chased from Aldrich Co. (USA). AnaeroPack Campylo was from
Mitsubishi Gas Chemical Co., Inc. (Japan). The anti-mouse Bax,
Bcl-2, procaspase-3 and b-actin antibodies were purchased from
Santa Cruz Biotechnology (USA). The enhanced chemilumines-
cence (ECL) Western blot system was from Pierce Co. (USA).
Bacterial strains
HP strains ATCC 49 503 and ATCC 43 504 were purchased from
American Type Culture Collection (USA). HPs were inoculated
into brucella agar plates supplemented with 7 % horse serum
and transferred into brucella broth containing 10 % FBS after 3
days. The bacteria were cultured for further 3 days at 37 8C in a
thermostatic rotary shaker under microaerophilic conditions
(AnaeroPack Campylo: 85 % N2, 10 % CO2 and 5 % O2).
198 Isolation of VacA toxin from HP
VacA was purified according to the modified method of Cover
and Blaser [13]. HP ATCC 49 503 was used as the source of toxin
purification. HP was cultured for 72 h at 37 8C in brucella broth
containing 10 % FBS in an ambient atmosphere containing 5 %
oxygen. The culture was centrifuged at 6,000 ” g for 30 min, and
proteins present in the supernatant were precipitated with a 50 %
saturated solution of ammonium sulfate. After centrifugation at
10,000 ” g for 40 min, the pellet was resuspended in 60 mM Tris
(pH 7.5). Hydrophobic interactive chromatography was per-
formed on a butyl-toyoperal (Sigma Co., U.S.A.) column (1 ” 5
cm) with the same buffer containing 0.6 M ammonium sulfate
and eluted with the same buffer containing 0.4 M ammonium
sulfate. The vacuolation-active fractions were dialyzed against
the same buffer.
Assay of cell viability
HeLa cells were cultured as a monolayer in plastic flasks in Dul-
becco's modified Eagle's medium (DMEM) containing 10 % FBS,
1 % antibiotic-antimycological solution and 3.5 g/L sodium bicar-
bonate under 5 % CO2 at 37 8C. Attached cells were released with
trypsin/EDTA and seeded at a density of 7.0 ” 103 cells/well in 96-
well tissue culture plates one day before the experiments.
Seeded HeLa cells were incubated with partially purified VacA
(0.05 mg/mL) for various times in a microtiter assay. Mitochon-
drial activity (cytotoxicity) of VacA toxin in HeLa cells was meas-
ured by the methylthiazolyldiphenyltetrazolium bromide (MTT)
Shin J-E et al. In Vitro Inhibitory ¼ Planta Med 2005; 71: 197 ± 201
assay [14]. The assay was not affected by the purified VacA toxin,
which did not show antioxidant activity. All assays were per-
formed in triplicate.
Immunoblot analysis
Immunoblot assays for procaspase-3, Bcl-2 and Bax protein ex-
pression levels were performed according to the method of Mae-
da et al. [7]. Cells were suspended in 50 mM Tris HCl (pH 7.4) buf-
fer containing 1 mM EGTA, 2 mM dithiothreitol, 25 mM sodium
b-glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride and
10 mg/mL aprotinin. An equal amount of protein extracts was
fractionated by sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis and electrophotometrically transferred to a polyvi-
nylidine fluoride membrane. The membrane was probed with
the antibodies described above. An ECL detection assay was per-
formed according to the manufacturer's instructions.
Assay of HP VacA vacuolation-inhibitory activity
HeLa cells were cultured as a monolayer in plastic flasks in
DMEM containing 10 % FBS, 1 % antibiotic-antimycological solu-
Downloaded by: University of Pittsburgh. Copyrighted material.
tion and 3.5 g/L sodium bicarbonate under 5 % CO2 at 37 8C. At-
tached cells were released with trypsin/EDTA and seeded at a
density of 7.0 ” 103 cells/well in 96-well tissue culture plates one
day before experiments.
The inhibitory effect of flavonoids on VacA vacuolation in HeLa
cells was measured by the neutral red uptake assay [3]. Briefly,
seeded HeLa cells were incubated for 16 h with HP-cultured
VacA toxin (0.5 mg/mL) or partially purified toxin (0.05 mg) and
serial dilutions of samples in a microtiter assay. To detect the va-
cuoles, cells were incubated for 8 min at room temperature with
100 mL of 0.05 % neutral red in phosphate-buffered saline (PBS)
and washed twice with 0.9 % NaCl containing 0.1 % BSA. After
the addition of 100 mL of acidified ethanol solution (70 % ethanol,
0.36 % HCl), the optical density of the extracted neutral red was
measured at 540 nm using a microtiter plate reader (Molecular
Devices, USA). All assays were performed in triplicate.
Inhibition assay of HP growth and urease activity
To evaluate HP ATCC 43 504 growth inhibition of flavonoids,
these compounds were two-fold diluted serially in brucella agar
supplemented with 7 % horse serum and their minimum inhibi-
tory concentrations (MICs) were measured according to the re-
ported method [15].
The activity of urease partially purified from HP ATCC 43504 cells
according to the previous method [10] was assayed at 37 8C by
measuring the amounts of ammonia per minute of incubation
periods from urea [16].
Assay of HP infection-inhibitory activity
KATO III cells were cultured in RPMI 1640 medium supplement-
ed with 10 % FBS, 1 % antibiotic-antimycological solution and
2.2 g/L sodium bicarbonate under 5 % CO2 at 37 8C. The cells
were harvested with trypsin/EDTA for the bacterial adhesion ex-
periment [17]. Serial dilutions of samples were incubated with
the equal volume of HP suspension in PBS for 30 min in a 37 8C
water bath and mixed with KATO III cells (5.0 ” 106 cells/mL).
After further 1 h incubation, the incubation mixture was loaded
on 15 % sucrose, centrifuged and washed once in PBS. Subse-
quently, the infected HP in KATO III cells was determined by
measuring urease activity of the precipitated cells [16]. Table 2 Inhibitory effects of flavonoids on VacA toxin vacuolation and
urease activity of HP
Statistical analysis
Agent IC50 (mM)
All the data from the experiment were expressed as mean 
VacA toxin vacuolation Urease activity
standard deviation and the statistical significance was deter-
mined using Student's t-test.
Epicatechina > 0.2 > 0.2
Epigallocatechin > 0.2 > 0.2
Epicatechin gallate > 0.2 > 0.2
Results
Epigallocatechin gallate > 0.2 > 0.2
Rutin > 0.4 > 0.2
To evaluate anti-HP activity of flavonoids, their inhibitory activ-
Quercitrin > 0.4 > 0.2
ities against HP growth and infection were measured (Table 1). Quercetin 0.046 > 0.2
Catechins, quercetin and naringenin weakly inhibited HP Naringin > 0.4 > 0.2

Original Paper
growth. Amongst the catechins, epigallocatechin gallate exhib- Naringenin 0.360 > 0.2
ited the most potent growth inhibition: its MIC value was 25 mg/ Daidzin > 0.4 > 0.2
mL. However, all tested flavonoids did not inhibit the HP infec- Daidzein > 0.4 > 0.2
tion into KATO III cells. Baicalin > 0.4 > 0.2
Baicalein > 0.4 > 0.2
The effects of flavonoids on HP vacuolation in HeLa cells by VacA Acetohydroxamic acid ±b 0.001

Downloaded by: University of Pittsburgh. Copyrighted material.

toxin and urease activity were investigated (Table 2). Quercetin
HP ATCC 43 504 and HP ATCC 49 504 were used for inhibitory assays of urease activity and
and naringenin inhibited VacA vacuolation of HeLa cells with IC50 vacuolation, respectively. The assays are described in Materials and Methods.
values of 0.046 and 0.36 mM, respectively. However, catechins a Inhibitory effects of catechins at less than 0.2 mM were determined, because these com-

pounds exhibited potent cytotoxicity.

and the other flavonoids tested did not inhibit HP vacuolation of b
Not determined.
HeLa cells. All tested compounds did not inhibit HP urease activity.

To understand the inhibitory mechanism of quercetin against HP
VacA vacuolation, VacA toxin was partially purified from HP
49 503 by ammonium sulfate precipitation and Q-Sepharose col-
umn chromatography, and the cytotoxicity of VacA toxin against
HeLa cells was measured (Fig. 1). Its cytotoxicity increased with
increasing incubation time. When the inhibitory activity of quer-
cetin on VacA vacuolation in HeLa cells was measured, quercetin
dose-dependently inhibited the vacuolation (Fig. 2). However, its
glycoside rutin did not inhibit the VacA toxin vacuolation (Fig. 3).
Therefore, we measured the inhibitory effect of quercetin on the
apoptosis of HeLa cells by VacA toxin (Fig. 4). VacA toxin activated
procaspase-3 to caspase-3 in HeLa cells. Quercetin at concentra-
tions of 0.0375 and 0.075 mM potently inhibited the activation of
procaspase-3 to caspase-3 at 16 h after VacA toxin treatment.
However, VacA toxin did not affect Bax and Bcl-2 protein levels in
HeLa cells. Quercetin also did not affect these protein levels. 199
Discussion

Flavonoids exhibit anti-inflammatory, anticancer, and antioxi-

Table 1 Inhibitory effects of flavonoids on the growth and infection of dant activities [10], [11], [18]. Recently it has been reported that
HP

Agent MICa IC50 of infection

(mg/mL) (mM)

Epicatechin 0.100 > 0.2

Epigallocatechin 0.050 > 0.2
Epicatechin gallate 0.050 > 0.2
Epigallocatechin gallate 0.025 > 0.2
Rutin > 0.1 > 0.2
Quercitrin > 0.1 > 0.2
Quercetin 0.100 > 0.2
Naringin > 0.1 > 0.2
Naringenin 0.100 > 0.2
Daidzin > 0.1 > 0.2
Daidzein > 0.1 > 0.2
Baicalin > 0.1 > 0.2
Baicalein > 0.1 > 0.2
Ampicillin 0.001 ±b Fig. 1 Cytotoxicity of HP VacA toxin (0.05 mg). Cell viability was as-
Heparin ± 0.022 sayed by MTT assay as described in Materials and Methods. Results
are expressed as percentages of viable cells. All values are means 
a
HP ATCC 43 504 was used for assays of MIC and inhibition of infection. S.D. (n = 3). * Significantly different from normal control group (p <
b
Not determined. 0.05).

Shin J-E et al. In Vitro Inhibitory ¼ Planta Med 2005; 71: 197 ± 201
 first investigated the VacA vacuolation-inhibitory effect of the
antioxidant agents ascorbic acid, glutathione, epicatechin and
trolox. However, these antioxidant agents did not exhibit antiva-
cuolation activity (data not shown). These compounds in the ex-
perimental conditions of the present study, except for catechins,
did not affect cell viability. Secondly, the effect of VacA toxin on
the production of nitric oxide from HeLa cells was examined, and
then a glutathione content assay was performed by an enzymatic
recycling technique. However, no significant change of gluta-
thione content by VacA was observed (data not shown).

Apoptosis is characterized by plasma membrane blebbing,

shrinkage, chromatin condensation, chromosomal DNA frag-
mentation, and the formation of membrane-bound apoptotic
Original Paper

bodies [6], [21]. Two major pathways leading to apoptosis have

been described. One pathway involves apoptosis mediated by
death receptors, such as CD95 (Fas) and tumor necrosis factor re-
ceptors. It processes effector caspases in the last step. In the
other pathway, various proapoptotic signals converge at the mi-
tochondria, provoking translocation of cytochrome c from the

Downloaded by: University of Pittsburgh. Copyrighted material.

mitochondria to the cytoplasm. Bcl-2 family members are asso-
ciated with mitochondria-related apoptosis. While the cell survi-
val-promoting molecules Bcl-2 and Bcl-X, localized at the outer
mitochondrial membrane, prevent translocation of cytochrome

Fig. 2 Inhibition of quercetin on VacA toxin-Induced vacuolation in

HeLa cells. HeLa cells were cultured in DMEM containing 10 % FBS.
VacA toxin (0.05 mg), which was partially purified from HP as de-
scribed in Materials and Methods, and each sample (0.075 and 0.15
mM) was treated in HeLa cells, incubated for 16 h at 37 8C and then
200 stained with neutral red. (A) treated without VacA toxin; (B) treated
with VacA toxin alone; (C), treated without VacA toxin, and stained
with neutral red; (D) treated with VacA toxin, and stained with neutral
red; (E), treated with VacA toxin and 0.075 mM quercetin, and stained
with neutral red; (F), treated with VacA toxin and 0.15 mM rutin, and
stained with neutral red. Arrow indicates vacuolations in HeLa.

Fig. 3 Inhibitory effects of quercetin and rutin on neutral red dye up-
take by VacA toxin-treated HeLa cells. HeLa cells were incubated with
these compounds show inhibition against HP growth [7], [8], [9], VacA toxin and test compounds, and stained with 0.05 % neutral red
[19], [20]. However, the effect of the flavonoids on infection and solution. Quercetin was used at the concentrations of 37.5, 75 and
vacuolation of HP have not been thoroughly studied. Therefore, 150 mM, and rutin was at the concentrations of 37.5 and 150 mM. All
values are means  S.D. (n = 3). # Significantly different from normal
the present study examined these compounds for their ability
control group (p < 0.05). * Significantly different from VacA toxin-treat-
to exhibit HP growth-inhibitory activity, and investigated in ed group (p < 0.05).
vitro their inhibitory effects on the growth and vacuolation of
HP VacA toxin as well as the infective properties of HP against
HeLa cells. Fig. 4 Inhibitory ef-
fect of quercetin on
Among the tested catechins, epigallocatechin gallate showed the the activation of
most potent inhibition of HP growth, followed by epigallocatechin, procaspase-3, Bax
and Bcl-2 of HeLa
epicatechin gallate, epicatechin, quercetin and naringenin as in
cells by VacA toxin.
the previous reports. However, other flavonoids did not show any Lane 1, normal con-
inhibition of HP growth. All tested flavonoids did not inhibit HP in- trol (HeLa cells treat-
fection into KATO III cells as well as HP urease activity. ed without VacA tox-
in); lane 2, HeLa cells
treated with VacA
Among the tested flavonoids, quercetin and naringenin inhibited
toxin (0.05 mg/mL);
HP vacuolation into HeLa cells. Quercetin showed the most po- lane 3, HeLa cells treated with VacA toxin (0.05 mg/mL) and quercetin
tent inhibition, with an IC50 value of 0.046 mM. It has been re- (0.075 mM); lane 4, HeLa cells treated with VacA toxin (0.05 mg/mL)
ported that quercetin exhibits antioxidant action. Therefore, we and quercetin (0.0375 mM).

Shin J-E et al. In Vitro Inhibitory ¼ Planta Med 2005; 71: 197 ± 201
c from the mitochondria, induced expression or enforced dimer-
ization of Bax results in mitochondrial dysfunction leading to cy-
tochrome c release. Jones et al. [5], Maeda et al. [7], and Kim et al.
[22] demonstrated that HP-induced apoptosis is related to cas-
pase-3 activity. Therefore, to investigate the apoptotic effect of
VacA in HeLa cells, we measured the procaspase-3 level in HeLa
cells with and without VacA treatment. VacA toxin significantly
decreased the procaspase-3 protein level. The decrease of pro-
caspase-3 protein seems to originate from the activation of pro-
caspase-3 to caspase-3 like the previous report of Maeda et al.
[7]. The antivacuolating agent quercetin potently inhibited the
activation of procaspase-3 protein induced by VacA. Neverthe-
less, no significant change was detected in the expression of
Bcl-2 and Bax. These results suggest that HP VacA toxin may in-
duce cell death transmitted via the proteolytic activation of a
cascade of caspases and that quercetin can inhibit the apoptotic
signaling.
Based on these findings, we believe that the antivacuolating
agent quercetin may improve gastric cell death induced by HP.
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