Journal of Ethnopharmacology 151 (2014) 1031–1039

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Apigenin has anti-atrophic gastritis and anti-gastric cancer progression

effects in Helicobacter pylori-infected Mongolian gerbils
Chao-Hung Kuo a,b, Bi-Chuang Weng a,c, Chun-Chieh Wu d, Sheau-Fang Yang d,e,
Deng-Chang Wu a,b,f,n, Yuan-Chuen Wang c,nn
a
Division of Gastroenterology, Department of Internal Medicine and Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC
b
Department of Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC
c
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC
d
Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan, ROC
e
Department of Pathology, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung, Taiwan, ROC
f
Division of Internal Medicine, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan, ROC

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: Apigenin, one of the most common flavonoids, is abundant in celery,
Received 6 September 2013 parsley, chamomile, passionflower, and other vegetables and fruits. Celery is recognized as a medicinal
Received in revised form vegetable in Oriental countries to traditionally treat inflammation, swelling, blood pressure, serum lipid,
19 November 2013
and toothache. In this study, we investigated apigenin treatment effects on Helicobacter pylori-induced
Accepted 20 November 2013
atrophic gastritis and gastric cancer progression in Mongolian gerbils.
Available online 25 December 2013
Materials and methods: Five to eight-week-old Mongolian gerbils were inoculated with Helicobacter
Keywords: pylori for four weeks without (atrophic gastritis group) or with N0 -methyl-N0 -nitro-N-nitroso-guanidine
Histological observation (MNNG) (gastric cancer group) in drinking water, and were then rested for two weeks. During the 7th–
Sydney system
32th (atrophic gastritis group) or the 7th–52th (gastric cancer group) weeks, they were given various
40 ,5,7-trihydroxyflavone
doses (0–60 mg/kgbw/day) of apigenin. At the end of the 32th (atrophic gastritis group) or the 52th
Anti-inflammation
(atrophic gastritis group) week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method.
The histological changes of Helicobacter pylori colonization, neutrophil and monocyte infiltrations, and
atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils were examined using
immunohistochemistry stain and Sydney System scoring.
Results: Apigenin treatments (30–60 mg/kgbw/day) effectively decreased atrophic gastritis (atrophic
gastritis group) and dysplasia/gastric cancer (gastric cancer group) rates in Mongolian gerbils. Apigenin
treatment (60 mg/kgbw/day) significantly decreased Helicobacter pylori colonization and Helicobacter
pylori-induced histological changes of neutrophil and monocyte infiltrations and atrophic gastritis in
both atrophic gastritis and gastric cancer Mongolian gerbils.
Conclusions: Apigenin has the remarkable ability to inhibit Helicobacter pylori-induced atrophic gastritis
and gastric cancer progression as well as possessing potent anti-gastric cancer activity.
& 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
More than 50% of the world population is infected with
Helicobacter pylori. Ten to fifty percent of infected individuals
n dominated gastritis with gastric atrophy and intestinal metaplasia,
Corresponding author at: Division of Gastroenterology, Department of Internal
Medicine and Cancer Center, Division of Internal Medicine, Kaohsiung Municipal
Hsiao-Kang Hospital, Kaohsiung Medical University Hospital; Department of
Medicine, Faculty of Medicine, College of Medicine, Kaohsiung Medical University;
Kaohsiung, Taiwan, ROC. Tel.: þ886 7 312 1101x7755.
nn
Corresponding author at: Department of Food Science and Biotechnology,
National Chung Hsing University, 250 Kuo-Kuang Rd., Taichung 402, Taiwan, ROC.
Tel: +886 4 2284 0385 x 4220; fax: +886 4 2285 4053.
E-mail addresses: dechwu@yahoo.com (ycwang@nchu.edu.tw (Y.-C. Wang).
develop peptic ulcer disease, and 1–3% of them progress to gastric
cancer (Taylor and Parsonnet, 1995). The WHO classified
Helicobacter pylori as a group I carcinogen in 1994 (Anonymous,
1994). Gastric adenocarcinomas are classified as either well
differentiated (known as intestinal-type) or undifferentiated
(known as diffuse-type); the former is characterized by a corpus-
whereas the latter is characterized by gastritis throughout the
stomach but no atrophy (Konturek et al., 2006; Fox and Wang,
2007).
The Correa pathway (Correa, 1992) defined intestinal-type ade-
nocarcinoma development as being a multistep and multifactor
process. Helicobacter pylori infection induces chronic inflammation
of the gastric mucosa, and then progresses to atrophic gastritis,

0378-8741/$ - see front matter & 2013 Elsevier Ireland Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jep.2013.11.040
1032 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039
intestinal metaplasia, dysplasia, and finally adenocarcinoma. Atrophic
gastritis appears to be a critical initiating step in the progression
toward gastric cancer (Correa, 1992; Fox and Wang, 2001; Fox and
Wang, 2007). Interestingly, higher gastric pH, nitrates, and salts are
stimulators, whereas ascorbic acid and β-carotene are inhibitors
(Correa, 1992; Fox and Wang, 2007).
Parietal, mucous neck, and chief cells are the major compo-
nents of the oxyntic gland of the stomach body, which are
responsible for gastric juice, mucus, and enzyme secretions,
respectively (Fox and Wang, 2007). Once Helicobacter pylori
attaches to host cells, the immune response immediately initiates
through cellular signal transduction cascade. Specifically in inflam-
mation defense, monocytes and polymorphonuclear neutrophils
infiltrate into oxyntic glands extensively triggering a cytokine
cascade that culminates in gland dilation and mineralization,
progressing to focal fibrosis and finally complete loss of oxyntic
parietal and chief cells (atrophic gastritis). (Fox and Wang, 2001;
Houghton et al., 2002; Konturek et al., 2006; Fox and Wang, 2007;
Konturek et al., 2009; Peek et al., 2010).
Apigenin (Fig. 1) is one of the most common flavonoids. It is
found in vegetables and fruits, and especially abundant in celery,
parsley, guava, bilimbi fruit, garlic, bell pepper, onion, chamomile,
and passionflower (Miean and Mohamed, 2001; Shukla and Gupta,
2010; Anonymous, 2013). Celery is recognized as a medicinal
vegetable in Oriental countries to traditionally treat inflammation
(pneumonia, cystitis, urethritis, pharyngolaryngitis, pharyngitis,
hepatitis, and nephritis), swelling, blood pressure, serum lipid, and
toothache (Lee, 1994). Chamomile prepared from the dried flowers
of Matricaria chamomilla is traditionally consumed as single
ingredient herbal tea in the America and European countries as
a natural aid for both sleeplessness and anxiety. Infusion chamo-
mile contains apigenin at 0.8–1.2% concentration (Shukla and
Gupta, 2010). Passionflower (Passiflora incarnata) was traditionally
used in the America and European countries for the treatment of
boils, wounds, liver problems, anxiety, insomnia, asthma, shingles,
and Parkinson0 s disease. Passionflower contains high levels of
flavonoids (apigenin, luteolin, and scopoletin) and indole alkaloids
(Anonymous, 2013).
A few papers have characterized apigenin0 s anti-inflammatory
activity both in vitro and in vivo. The relevant mechanisms include:
(1) suppression of nuclear factor kappa B (NF-κB) activation; and
(2) inhibitions of cyclooxygenase-2 (COX-2), inducible nitric oxide
synthase (iNOS), and proinflammatory cytokine [interleukin IL-1β
(IL-1β), IL-6, IL-8, and tumor necrosis factor-α (TNF-α)] production
(Raso et al., 2001; Smolinski and Pestka, 2003; Ueda et al., 2004;
Comalada et al., 2006; Lee et al., 2007; Nicholas et al., 2007; Ha
et al., 2008). Anti-gastric carcinoma activity of apigenin was also
reported being due to its anti-proliferation and apoptosis-inducing
activities (Wu et al., 2005; Yuan et al., 2007; Li-Weber, 2013).
Additionally, other bioactivities including anti-platelet aggrega-
tion, antioxidation, and anti-Helicobacter pylori activity have been
also studied (Zhang et al., 2008; Han et al., 2009; Das et al., 2010;
Wright et al., 2010). The bioactivity of apigenin is probably due to
hydroxylation at A and B rings coupled with a double bond at C2
and C3 in the flavonoid skeleton (Kawaii et al., 1999; Raso et al.,
2001; Ueda et al., 2004; Comalada et al., 2006), and its intrinsic
scavenging property (Wang and Huang, 2013).
Fig. 1. Chemical structure of apigenin (40 ,5,7-Trihydroxyflavone).
In our previous paper (Wang and Huang, 2013) with an in vitro
focus, remarkable anti-Helicobacter pylori induced inflammation
was demonstrated in apigenin, in which the suppression of NF-κB
pathway activation and inhibition of inflammatory factor [reactive
oxygen species (ROS), COX-2, intercellular adhesion molecule-1
(ICAM-1), IL-6, and IL-8] expressions were involved. Consequently,
in this study, we investigated apigenin treatment effects on
Helicobacter pylori-induced atrophic gastritis and gastric cancer
progression in Mongolian gerbils using immunohistochemistry
stain and Sydney System scoring.
2. Materials and methods
2.1. Reagents
Apigenin (98% purity) was purchased from Shaanxi Huike
Botanical Development Co. (Shaanxi, China), with ethanol and
dimethyl benzene from JT Baker (USA), fetal bovine serum (FBS)
from SAFC Biosciences (Australia), Brucella broth from Becton
Dickinson and Company (USA), basal granular diet from Altromin
Spezialfutter GmbH & Co. (Germany), Embedding Medium from
Leica Biosystems (Germany), N0 -methyl-N0 -nitro-N-nitroso-guani-
dine (MNNG) from Tokyo Chemical Industry Co., Ltd. (Tokyo,
Japan), methyl cellulose from Nacalai Tesque, Inc. (Kyoto, Japan),
Helicobacter pylori Rapid Test Device from ABON Biopharm Co., Ltd.
(China), normal saline from Nang Kuang Pharmaceutical Co., Ltd.
(Tainan, Taiwan), neutral-buffered formalin from Hsiang Hsin
(Taipei, Taiwan), and hematoxylin-eosin (H-E) from Surgipath
(USA).
2.2. Helicobacter pylori strain
Helicobacter pylori strain KMUH-G926 (cagA þ/vacAþ ) was
isolated from a Helicobacter pylori-severely infected Mongolian
gerbil. A volume of 0.1 mL of Helicobacter pylori suspension was
added to 9 mL containing 10% (v/v) FBS of Brucella broth, and then
was cultivated in an Anaerobic Workstation (Ruskinn Bugbox/
BB1103346, England) with 5% CO2 and 10% CO2-in-air at 37 1C for
72 h to produce 9  108 CFU/mL of the bacterial counts.
2.3. Mongolian gerbils and experimental designs
Five to eight-week-old male and female Mongolian gerbils (50–
55 g weights) were obtained from the Laboratory Animal Center of
Taiwan University (Taipei, ROC), and housed in an air-conditioned
biohazard room with a 12:12-h light/dark cycle, 70–80% humidity,
and 22–24 1C temperature. They were given water and basal
granular diet ad libitum. Experiments were performed according
to the experimental guidelines of the ethics committee of Kaoh-
siung Medical University Chung-Ho Memorial Hospital Laboratory
Animal Center. Those guidelines were established according to the
Guide for The Care and Use of Laboratory Animals by the American
National Research Council (NRC) and were approved by the ethics
committee constituted in accordance with the rules and guidelines
stated by government of Taiwan (Article 16 of the Animal Protec-
tion Act announced on 03 Jun, 2010; Document no. 0990041068).
The experiments were divided into atrophic gastritis and gastric
cancer groups. For the atrophic gastritis group (Fig. 2), Mongolian
gerbils were divided into six groups, with 10 Mongolian gerbils in
each group. Group I-A was the vehicle. In the first four weeks,
groups I-C–I-F were orally inoculated with Helicobacter pylori
(9  108 CFU/ml, 2 mL) twice weekly and groups I-A–I-B were only
given Brucella broth. During the 7th to the 32nd weeks, groups
I-A I-F were intragastrically administered various doses (0–60 mg/
kgbw/day) of apigenin once daily in which apigenin was suspended
 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039
Fig. 2. Experimental design for Mongolian gerbils with apigenin treatments against
Helicobacter pylori-induced atrophic gastritis. Mongolian gerbils (males and
females, 50–55 g weights) were divided into six groups with 10 Mongolian gerbils
in each group. Group I-A was the vehicle. In the first four weeks, groups I-C–I-F
were orally inoculated with Helicobacter pylori (9  108 CFU/mL, 2 mL) twice
weekly and groups I-A–I-B were only given Brucella broth. During the 7th–32nd
weeks, groups I-A–I-F were intragastrically administered various doses (0–60 mg/
kgbw/day) of apigenin once daily in which apigenin was suspended in 0.5% methyl
cellulose. At the end of the 32nd week, all Mongolian gerbils were sacrificed using
the CO2 asphyxia method.
Fig. 3. Experimental design for Mongolian gerbils with apigenin treatments against
Helicobacter pylori-induced gastric cancer. Mongolian gerbils (males and females,
50–55 g weights) were divided into seven groups with eight Mongolian gerbils in
each group. Group II-A was the vehicle. In the first four weeks, groups II-D  II-G
were orally inoculated with Helicobacter pylori (9  108 CFU/mL, 2 mL) twice
weekly and groups II-A–II-C were only given Brucella broth. During the 2nd–20th
weeks, groups II-B–II-G were given 50 μg/mL MNNG with drinking water, and then
switched to distilled water. Additionally, at the 7th week, groups II-A–II-G were
intragastrically administered various doses (0–60 mg/kgbw/day) of apigenin once
daily until the end of the 52nd week in which apigenin was suspended in 0.5%
methyl cellulose. At the end of the 52nd week, all Mongolian gerbils were sacrificed
using the CO2 asphyxia method.
in 0.5% methyl cellulose. At the end of the 32nd week, all Mongolian
gerbils in the atrophic gastritis group were sacrificed using the CO2
asphyxia method. For the gastric cancer group (Fig. 3), Mongolian
gerbils were divided into seven groups, with eight Mongolian
gerbils in each group. Group II-A was the vehicle. In the first four
weeks, groups II-D–II-G were orally inoculated with Helicobacter
pylori (9  108 CFU/mL, 2 mL) twice weekly and groups II-A–II-C
were only given Brucella broth. During the 2nd to the 20th weeks,
1033
groups II-B–II-G were given 50 μg/mL MNNG with drinking water,
and consequently switched to distilled water. Additionally, at the
7th week, groups II-A–II-G were intragastrically administered
various doses (0–60 mg/kgbw/day) of apigenin once daily until
the end of the 52nd week in which apigenin was suspended in 0.5%
methyl cellulose. At the end of the 52nd week, all Mongolian gerbils
in the gastric cancer group were sacrificed using the CO2 asphyxia
method.
2.4. Body and relative organ weights
After sacrificing, the body and organs (stomach, liver, and
kidneys) of each Mongolian gerbil were weighed. Relative weight
(%) was calculated as the absolute weight of each organ compared
to the individual body weight.
2.5. Helicobacter pyloriHelicobacter pyloriinfection assay
The fresh heart blood of the sacrificed Mongolian gerbils was
collected and centrifugated at 1308g for 10 min. A volume of 30 μL
of the supernatant was examined for Helicobacter pylori infection
using an Helicobacter pylori Rapid Test Device.
2.6. Tissue preparation and histological examination
The Mongolian gerbil stomachs were dissected along the
greater curvature, washed with normal saline, fixed with 10%
neutral-buffered formalin for 24 h, dehydrated with ethanol and
dimethyl benzene; embedded in Embedding Medium, and then
sliced into 4 μm-thick sections.
The sections were stained with H-E, and then histologically
examined for Helicobacter pylori density, neutrophil and monocyte
infiltrations, intestinal metaplasia, and dysplasia under a light
microscope (Olympus DP72, Tokyo, Japan) at 1000 times magni-
fication, and at 40x for atrophic gastritis and gastric cancer. The
degree of Helicobacter pylori colonization, infiltrations in neutro-
phils and monocytes, and atrophic gastritis were evaluated using
the Sydney System (Dixon et al., 1996) on the basis of a four-point
scale (0–3, 0¼ normal; 1¼ mild; 2, moderate; 3 ¼ marked). The
dysplasia was identified as nuclear abnormalities including increased
size, hyperchromatism, and an irregular shape of the glandular
epithelial cells; whereas the gastric cancer was identified as cells
forming irregular, invasive neoplastic glands with destruction of the
normal gastric mucosa and submucosa architecture.
2.7. Statistical analysis
Data for body and relative organ weights, Sydney System scores
for Helicobacter pylori colonization, infiltrations in neutrophils and
monocytes, and atrophic gastritis were subjected to one-way
analysis of variance (one-way ANOVA) with Dunnett0 s test by SPSS
10.0 software (SPSS Inc., USA) using least significant difference to
identify significant differences of experimental groups from the
control. Po 0.05 or P o0.01 was considered to be significant.
3. Results
3.1. Body and relative organ weights
For the atrophic gastritis group (Table 1), neither the Helico-
bacter pylori infection (I-C) nor the 60 mg apigenin/kgbw/day
treatment (I-B) resulted in significant differences of the body
and relative organ (stomach, liver, and kidney) weights from the
vehicle (I-A) (P o0.05). There were no significant differences
in those weights between the 10–60 mg/kgbw/day of apigenin
1034 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039

Table 1 cancer (II-D–II-F treatments, 8/8) groups. 100% (10/10) of atrophic

Body weights and relative organ weights of Helicobacter pylori-induced atrophic gastritis rates were found in both the I-C and II-D treatments (both
gastritis and gastric cancer in Mongolian gerbils treated with various doses
being þHelicobacter pylori/–apigenin); 30 and 60 mg/kgbw/day of
(0–60 mg/kgbw/day) of apigenin.
apigenin treatments (I-E and I-F) decreased those rates to 80% and
Treatment Body weight (g) Relative organ weight (%) 40%, respectively, in the atrophic gastritis group treatments, but
not for the gastric cancer group. Intestinal metaplasia was not
Stomach Liver Kidney observed in either group. Both dysplasia and gastric cancer
Atrophic gastritis group
histological changes were not found in the atrophic gastritis
I-A 70.17 2.1 0.92 7 0.03 3.20 7 0.08 0.747 0.04 group; however, they were discovered in the gastric cancer group
I-B 81.9 7 3.0 1.007 0.03 3.577 0.07 0.79 7 0.03 (II-D–II-G treatments). With increasing treatment doses (30 and
I-C 75.17 3.1 1.047 0.04 3.497 0.07 0.747 0.02 60 mg/kgbw/day, II-F and II-G), both rates decreased from 100%
I-D 73.17 3.1 1.017 0.03 3.477 0.11 0.82 7 0.02
and 88% (II-D) to 13–75% and 0–63%, respectively, for dysplasia
I-E 74.8 7 3.1 1.047 0.05 3.477 0.08 0.80 7 0.02
I-F 80.5 7 2.3 1.02 7 0.03 3.54 7 0.06 0.767 0.02 and gastric cancer.
Gastric cancer group Summarizing Table 2, Helicobacter pylori infection resulted in
II-A 78.0 7 2.7 1.02 7 0.03 3.577 0.17 0.86 7 0.04 atrophic gastritis in 32 week-treated Mongolian gerbils (the
II-B 80.6 7 3.8 0.84 7 0.02 3.54 7 0.12 0.89 7 0.02 atrophic gastritis group). Furthermore, Helicobacter pylori infection
II-C 78.0 7 3.9 0.92 7 0.05 3.28 7 0.09 0.86 7 0.03
II-D 71.5 7 2.4 1.05 7 0.06 3.75 7 0.10 0.83 7 0.02
with MNNG resulted in gastric cancer in 52 week-treated Mon-
II-E 72.1 7 2.9 1.147 0.12 3.517 0.12 0.80 7 0.03 golian gerbils (the gastric cancer group). However, 30 and 60 mg/
II-F 72.0 7 2.4 1.08 7 0.06 3.82 7 0.08 0.95 7 0.10 kgbw/day of apigenin treatments effectively reduced those inci-
II-G 75.6 7 2.9 1.017 0.05 3.78 7 0.15 0.87 7 0.03 dences in both groups.
Relative organ weight (%)¼ [absolute organ weight (g)/body weight of Mongolian
gerbils (g)]  100. Values presented are the mean 7 standard error, which derived 3.3. Apigenin effects on histological changes of atrophic gastritis
from 10 and 8 Mongolian gerbils for groups atrophic gastritis and gastric cancer, Mongolian gerbils
respectively. There are no significant difference from I-C and II-D for atrophic
gastritis and gastric cancer groups, respectively, at Po 0.05 (atrophic gastritis
Fig. 4(a–c) are histological changes under a light microscope,
group) and P o 0.01 (gastric cancer group).
and Fig. 4(d–f) are Sydney System scores of those changes.
As shown in Fig. 4(d–f), there were no histological changes
(Helicobacter pylori density, neutrophil and monocyte infiltrations,
Table 2 and atrophic gastritis) for the vehicle (I-A) and apigenin-only (I-B)
Helicobacter pylori infection and histological change ratesa of Helicobacter pylori- treatments; however, Helicobacter pylori infection (I-C) signifi-
induced atrophic gastritis and gastric cancer in Mongolian gerbils treated with cantly increased those changes [2.7070.48 (Helicobacter pylori
various doses (0–60 mg/kgbw/day) of apigenin. density) (F5, 54 ¼105.46, P o0.05; one-way ANOVA), 2.90 70.32
Treatment Helicobacter Atrophic Intestinal Dysplasia Gastric
(neutrophil) (F5, 54¼ 153.95, Po0.05; one-way ANOVA), 2.90 7
pylori infection gastritis metaplasia cancer 0.32 (monocyte) (F5, 54¼ 85.35, P o0.05; one-way ANOVA), and
1.00 70.00 (atrophic gastritis) (F5, 54 ¼29.52, P o0.05; one-way
Atrophic gastritis group ANOVA)]. In contrast, apigenin treatment (60 mg/kgbw/day) sig-
I-A 0 (0/10) 0 (0/10) 0 (0/10) 0 (0/10) 0 (0/10)
nificantly decreased those scores to 2.00 70.47, 1.90 70.57, 1.80 7
I-B 0 (0/10) 0 (0/10) 0 (0/10) 0 (0/10) 0 (0/10)
I-C 100 (10/10) 100 (10/10) 0 (0/10) 0 (0/10) 0 (0/10) 0.63, and 0.40 70.52, respectively (F5, 54 ¼105.46, 153.95, 85.35,
I-D 100 (10/10) 100 (10/10) 0 (0/10) 0 (0/10) 0 (0/10) and 29.52, respectively; P o0.05; one-way ANOVA).
I-E 100 (10/10) 80 (8/10) 0 (0/10) 0 (0/10) 0 (0/10)
I-F 100 (10/10) 40 (4/10) 0 (0/10) 0 (0/10) 0 (0/10)
3.4. Apigenin effects on histological changes of gastric cancer
Gastric cancer group
II-A 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8) Mongolian gerbils
II-B 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8)
II-C 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8) 0 (0/8) Fig. 5(a–c) are histological changes under a light microscope,
II-D 100 (8/8) 100 (8/8) 0 (0/8) 100 (8/8) 88 (7/8)
and Fig. 5(d–f) are Sydney System scores of those changes.
II-E 100 (8/8) 100 (8/8) 0 (0/8) 100 (8/8) 88 (7/8)
II-F 100 (8/8) 100 (8/8) 0 (0/8) 75 (6/8) 63 (5/8)
As shown in Fig. 5(d–f), there were no histological changes
II-G 100 (8/8) 100 (8/8) 0 (0/8) 13 (1/8) 0 (0/8) (Helicobacter pylori density, neutrophil and monocyte infiltrations;
and atrophic gastritis) for the vehicle (II-A), MNNG-only (II-B), and
a
the percentage of positive numbers to total test numbers. apigenin-only (II-C) treatments; however, Helicobacter pylori infection
(II-D) significantly increased those changes [2.8870.35 (Helicobacter
treatments (I-D-I–I-F) and the 0 mg/kgbw/day treatment (I-C) pylori density) (F6, 49¼147.74, Po0.05; one-way ANOVA), 2.8870.35
(P o0.05). (neutrophil) (F6, 49¼130.62, Po0.05; one-way ANOVA), 2.8870.35
For the gastric cancer group, the results were similar with the (monocyte) (F6, 49¼ 117.66, Po0.05; one-way ANOVA), and 3.007
atrophic gastritis group. The Helicobacter pylori infection (II-D), 0.00 (atrophic gastritis)] (Po0.05) (F6, 49¼51.57, Po0.05; one-way
50 μg/mL MNNG given (II-B), and 60 mg apigenin/kgbw/day treat- ANOVA). Contrarily, apigenin treatment (60 mg/kgbw/day) signifi-
ment (II-C) did not result in significant changes of the body and cantly decreased those scores to 1.8870.35, 1.7570.46, 2.1370.64,
relative organ weights from the vehicle (II-A) (P o0.01). There and 1.7570.34, respectively (F6, 49¼147.74, 130.62, 117.66, 51.57,
were no significant differences in those weights between the respectively; Po0.05; one-way ANOVA) (Po0.05).
0–60 mg apigenin/kgbw/day treatments (II-D–II-G) (Po 0.01).

3.2. Helicobacter pylori infection and histological change rates 4. Discussion

As shown in Table 2, in spite of apigenin treatments (10–60 mg In this study, Helicobacter pylori infection of Mongolian ger
apigenin/kgbw/day), 100% of Helicobacter pylori colonization rates bils resulted in atrophic gastritis and gastric cancer after 32 and
were found in all Helicobacter pylori-infected Mongolian gerbils for 52 week treatments, respectively, for which the histological
both the atrophic gastritis (I-C–I-F treatments, 10/10) and gastric changes (Helicobacter pylori colonization, neutrophil and monocyte
 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039 1035

I-A I-C I-F

4 4
neutrophil
monocyte
Sydney score of H. pylori density

neutrophil/monocyte infiltration

3 3
Sydney score of

2 2

1 1

0 0
H. pylori H. pylori
Apigenin 0 60 0 10 30 60 Apigenin 0 60 0 10 30 60
(mg/kgbw/day) (mg/kgbw/day)
I-A I-B I-C I-D I-E I-F I-A I-B I-C I-D I-E I-F

4
Sydney score of atrophic gastritis

0
H. pylori
Apigenin 0 60 0 10 30 60
(mg/kgbw/day)
I-A I-B I-C I-D I-E I-F
Fig. 4. Effect of apigenin treatments (0–60 mg/kgbw/day, I-A–F) on histological changes (a–c) and Sydney System scores (d–f) for Helicobacter pylori-induced atrophic
gastritis in Mongolian gerbils in which (a) and (d) are Helicobacter pylori density, (b) and (e) are neutrophil/monocyte infiltrations, and (c) and (f) are atrophic gastritis. The
black and red arrows in (b) indicate neutrophils and monocytes, respectively. The bars of Sydney scores (d–f) represented are the mean 7 standard error (n ¼10) with single
star showing the significant difference from treatment I-C ( þ Helicobacter pylori/-apigenin) at P o 0.05. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
1036 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039

II-A II-D II-G

neutrophil
monocyte
4
4
* *
*
Sydney scores of neutrophil
Sydney score of H. pylori

3
/monocyte infiltration

3
density

2
2

1
1

0 0
H. pylori H. pylori
MNNG MNNG
Apigenin 0 0 60 0 10 30 60 Apigenin 0 0 60 0 10 30 60
(mg/kgbw/day) (mg/kgbw/day)
II-A II-B II-C II-D II-E II-F II-G II-A II-B II-C II-D II-E II-F II-G

f 4
*
Sydney score of atrophic

3
gastritis

0
H. pylori
MNNG
Apigenin 0 0 60 0 10 30 60
(mg/kgbw/day)
II-A II-B II-C II-D II-E II-F II-G
Fig. 5. Effect of apigenin treatments (0–60 mg/kgbw/day, II-A–G) on histological changes (a–c) and Sydney System scores (d–f) for Helicobacter pylori-induced cancer in
Mongolian gerbils in which (a) and (d) are Helicobacter pylori density, (b) and (e) are neutrophil/monocyte infiltrations, and (c) and (f) are atrophic gastritis. The black and red
arrows in (b) indicate neutrophils and monocytes, respectively. The bars of Sydney scores (d–f) represented are the mean 7standard error (n¼ 8) with single star showing
the significant difference from treatment II-D ( þ Helicobacter pylori/-apigenin) at P o 0.05. (For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039 1037

infiltrations, atrophic gastritis, dysplasia, or gastric cancer) were
observed. Notably, apigenin treatment (60 mg/kgbw/day) effectively
decreased such changes either in the atrophic gastritis or gastric
cancer Mongolian gerbils.
In our previous in vitro study (Wang and Huang, 2013),
apigenin treatments significantly inhibited NF-κB activation in
the Helicobacter pylori-infected adenocarcinoma cells. Thus, the
downstream inflammatory factor [COX-2, ICAM-1, ROS, IL-6, IL-8,
and reactive oxygen species (ROS)] expressions remarkably
decreased. Additionally, the intrinsic radical scavenging property
of apigenin may contribute to ROS deletion (Zhang et al., 2008;
Han et al., 2009). In Table 2 and Fig. 4, the 60 mg/kgbw/day of
apigenin treatment effectively decreased neutrophil and monocyte
infiltrations and atrophic gastritis in the Helicobacter pylori-
infected Mongolian gerbils. Both in vitro and in vivo studies have
showed consistent results. We strongly suggested that apigenin
has an ability to inhibit extensive, Helicobacter pylori-induced
inflammation (superficial gastritis) and atrophic gastritis in Mon-
golian gerbils through the suppression of NF-κB activation and the
related inflammatory factor inhibition as well as the intrinsic free
radical scavenging property of apigenin.
Atrophic gastritis is a critical initiating step in the progression
toward gastric cancer (Correa, 1992; Houghton et al., 2002; Fox
and Wang, 2007). In Figs. 4 and 5, the Sydney scores of atrophic
gastritis for the atrophic gastritis group (1.007 0.00, I-C) were
much lower than that of the gastric cancer group (3.00 70.00,
II-D) because of MNNG incorporation and more than 20 week
treatment in gastric cancer group. Histological changes of dyspla-
sia and gastric cancer for the gastric cancer group were observed
at 100% and 88% rates, respectively (Table 2); importantly, dyspla-
sia is a necessary step for gastric cancer development (Correa,
1992; Houghton et al., 2002; Fox and Wang, 2007). Notably, the
60 mg/kgbw/day of apigenin treatment significantly reduced dys-
plasia and gastric cancer rates by 87 and 100%, respectively
(Table 2), and reduced the Sydney score of atrophic gastritis by
42% (Fig. 5). From this evidence, we can conclude that apigenin not
only significantly decreased atrophic gastritis incidence, but also
inhibited the progression of dysplasia and gastric cancer in the
Helicobacter pylori-infected Mongolian gerbils. Notably, apigenin
was also reported for its apoptosis-inducing activity in gastric
carcinoma cells through Akt activity inhibition (Wu et al., 2005;
Yuan et al., 2007). The gastric cancer reductions in Mongolian
gerbils in this study (Table 2 and Fig. 5) were partially due to the
suppression of Helicobacter pylori-induced inflammation, and thus
the gastric cancer progression was inhibited. Apigenin has a gastric
cancer prevention effect. Equally, those rate reductions were also
probably due to apoptosis in gastric adenocarcinoma in Mongolian
gerbils. In our previous study (Wang and Huang, 2013), 74 μM of
apigenin significantly decreased IκBα degradation and COX-2/ROS
production. The active NF-κB (dimer p50/p65) is a pro-apoptotic
gene (Bad, Bak, and Bax) inhibitor, which inhibits apoptosis in
gastric carcinoma cells (Noto and Peek, 2012; Li-Weber, 2013).
COX-2 inhibitors are able to inhibit some cell cycle checkpoints
and pro-apoptotic gene expressions (Sobolewski et al., 2010).
Moreover, ROS is a stimulator for NF-κB activation (Handa and
Yoshikawa, 2010; Li-Weber, 2013). Therefore, suppression of NF-κB
activation, reduction of COX-2/ROS production, and the intrinsic
scavenging property of apigenin observed in our previous study
(Wang and Huang, 2013) would be the key effectors to activate
pro-apoptotic gene expressions resulting in apoptosis in gastric
adenocarcinoma in Mongolian gerbils, and finally leading to
decreased gastric cancer rates (Table 2, Fig. 5). Apigenin has a
potent anti-gastric cancer effect. Furthermore, a few studies have
pointed out that the most significant anti-inflammatory effects of
flavonoids were demonstrated in hydroxylations at positions 5, 7,
30 and 40 coupled with a double bond at C2–C3 in the flavonoid
skeleton. Specifically, luteolin (30 ,40 ,5,7-tetrahydroxyflavone) and
quercetin (30 ,40 ,5,7-tetrahydroxyflavonol) had the greatest anti-
inflammatory activity and anti-TNF-α production among the test
flavonoids. Apigenin (40 ,5,7-trihydroxyflavone) is characterized by
3 hydroxylations at C-40 ,5,7 and a double bond at C2–C3 in the
flavonoid skeleton; the anti-inflammatory activity was slightly
lower than that of luteolin and quercetin but still remarkable
(Kawaii et al., 1999; Raso et al., 2001; Ueda et al., 2004; Comalada
et al., 2006). Those chemical structure features of apigenin can
further explain the good anti-inflammatory and anti-gastric cancer
progression/potent anti-gastric cancer activities demonstrated in
our previous (Wang and Huang, 2013) and present studies.
Herein, the role that apigenin played on the Correa pathway
(Correa, 1992; Fox and Wang, 2001) in Mongolian gerbils is proposed
in Fig. 6. Apigenin has the remarkable ability to inhibit the progres-
sion of Helicobacter pylori-induced superficial gastritis, atrophic
gastritis, dysplasia, and gastric cancer in Mongolian gerbils.
Few in vivo studies have been concerned with the anti-
Helicobacter pylori-inducing gastritis and gastric cancer of natural
products or clinical medicines. The natural products quercetin
(González-Segovia et al., 2008), caffeic acid phenethyl ester
(Toyoda et al., 2009), Cladosipbon fucoidan (Shibata et al., 2003),

6 weeks
32 weeks
52 weeks

H. pylori

Superficial Atrophic Gastric

Dysplasia
gastritis gastritis adenocarcinoma
Infected Mongolian gerbil

Apigenin

Fig. 6. Inhibitory effects of apigenin on the Correa pathway in Helicobacter pylori-infected Mongolian gerbils. Five to eight-week-old Mongolian gerbils were inoculated with
Helicobacter pylori for four weeks without (atrophic gastritis group) or with MNNG (gastric cancer group) in drinking water, and were then rested for two weeks. During the
7th–32th (atrophic gastritis group) or the 7th–52th (gastric cancer group) weeks, they were given various doses (0–60 mg/kgbw/day) of apigenin. At the end of the 32th
(atrophic gastritis group) or the 52th (atrophic gastritis group) week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method. From the results, apigenin
treatment (60 mg/kgbw/day) significantly inhibited the progression of superficial gastritis, atrophic gastric, dysplasia, and gastric adenocarcinoma in Mongolian gerbils.
1038 C.-H. Kuo et al. / Journal of Ethnopharmacology 151 (2014) 1031–1039
apple peel fraction (Pastene et al., 2010), Aloe vera DMSO extract
(Kumari et al., 2010), Japanese rice-fluid (Ishizone et al., 2007), 95%
ethanol extracts of finger-root and turmeric rhizomes (Mahady
et al., 2006), green tea extract (Matsubara et al., 2003), and garlic
water–ethanol extract (Iimuro et al., 2002) were reported for their
anti-inflammatory activity in Helicobacter pylori-infected Mongo-
lian gerbils, guinea pigs, or albino rats. Significant reductions in
the histological changes (neutrophil/monocyte infiltration, gastri-
tis, intestinal plasia, chronic/acute inflammation, and gastric
lesions) have been well discussed. However, none of these focused
on the anti-gastric cancer activity induced by Helicobacter pylori.
There were two clinical COX-2 inhibitors [etodolac (Magari et al.,
2005) and celecoxib (Kuo et al., 2009)] reported to have both anti-
gastritis and gastric cancer activities in Helicobacter pylori-infected
Mongolian gerbils. Specifically, comparing our study0 s apigenin
and quercetin (González-Segovia et al., 2008), both compounds
have similar chemical structures as aforementioned, and both
exhibited anti-Helicobacter pylori induced gastric inflammation in
test animals. However, the treatment dosage for apigenin (60 mg/
kg) was much lower than quercetin (200 mg/kg). Moreover, anti-
gastric inflammatory, anti-gastric cancer progression, and potent
anti-gastric cancer activities were demonstrated in apigenin;
contrastingly, only the anti-gastric inflammatory effect was
reported in quercetin (González-Segovia et al., 2008). In conclu-
sion, apigenin is the first natural product showing anti-gastritis,
anti-gastric cancer progression, and potent anti-gastric cancer
activities in Helicobacter pylori-infected test animals.
Acknowledgments
This work was supported by Grants from the Excellence for
Cancer Research Center, Department of Health, Executive Yuan,
Taiwan, ROC. (DOH101-TD-C-111-002), and the National Science
Council, Taiwan, ROC. (NSC 98-2313-B-005-017-MY3). We thank
Prof. Chien-Hung Lee, Department of Public Health, Kaohsiung
Medical University (Kaohsiung, Taiwan, ROC.) who kindly assisted
in the statistical analysis of this study.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2013.11.040.
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