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Keywords:

  • canolol;
  • antioxidant;
  • canola oil;
  • Helicobacter pylori;
  • Mongolian gerbils

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Oxidative stress is linked to gastric carcinogenesis because of its ability to damage DNA. Here we examined antioxidative and anti-inflammatory effects of 4-vinyl-2,6-dimethoxyphenol (canolol), a recently identified potent antioxidative compound obtained from crude canola oil, on Helicobacter (H.) pylori-induced gastritis and gastric carcinogenesis using a Mongolian gerbil model. The animals were allocated to H. pylori-infection alone (12 weeks) or H.pylori + N-methyl-N-nitrosourea (MNU) administration (52 weeks). After oral inoculation of H. pylori, they were fed for 10 and 44 weeks with or without 0.1% canolol. H. pylori-induced gastritis, 5′-bromo-2′-deoxyuridine (BrdU) labeling and scores for cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) immunohistochemistry were attenuated in the canolol-treated groups. Expression of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), COX-2 and iNOS mRNA in the gastric mucosa, and serum 8-hydroxy-2′-deoxyguanosine (8-OHdG), anti-H. pylori IgG and gastrin levels were also significantly lower in canolol-treated groups. Furthermore, the incidence of gastric adenocarcinomas was markedly reduced in the H. pylori + MNU + canolol-treated group [15.0% (6/40)] compared to the control group [39.4% (13/33)] (p < 0.05). These data indicate canolol to be effective for suppressing inflammation, gastric epithelial cell proliferation and gastric carcinogenesis in H. pylori-infected Mongolian gerbils. Interestingly, the viable H. pylori count was not changed by the canolol containing diet. Thus, the data point to the level of inflammation because of H. pylori rather than the existence of the bacteria as the determining factor. Importantly, canolol appears to suppress induction of mRNAs for inflammatory cytokines. © 2007 Wiley-Liss, Inc.

Helicobacter pylori (H. pylori) is now considered as the most important etiological agent for chronic gastritis and peptic ulcer disease, as well as a cause of gastric carcinoma.1, 2 There is accumulating evidence that eradication of H. pylori in the stomach by administration of oral antimicrobial agents results in the resolution of H. pylori-infected chronic active gastritis and peptic ulceration and significantly lowers the risk of stomach tumor development in patients without precancerous lesions.3, 4 However, such bacterial eradication treatment has not been always successful. The occurrence of antibiotic-resistant H. pylori has been reported, and it is occasionally associated with adverse effects.5 Therefore, it is still desirable to develop alternative approaches for cancer prevention and a number of studies have demonstrated protective effects of plant extracts against H. pylori infection, such as green tea catechins6 and a garlic extract.7 In a previous study, we also found fruit-juice concentrate of Japanese apricot (ume) (CJA) to decrease the number of H. pylori and suppress chronic active gastritis in gerbils,8 although the mechanism was unclear. H. pylori infection has been reported to cause production of H2O2 in AGS human gastric epithelial cells, which might contribute to carcinogenesis.9 Correa et al. reported antioxidants like vitamin C or vitamin E have protective effects against H. pylori-induced lesions due to their free radical scavenging activity.10, 11

4-Vinyl-2,6-dimethoxyphenol (canolol), was recently identified as a potent antioxidative compound in crude canola oil, exhibiting more potent antialkylperoxyl [ROO] radical activity than well-known antioxidants, like α-tocopherol, vitamin C, β-carotene, rutin and quercetin.12 We previously reported strong scavenging capacity against the endogenous mutagen, peroxynitrite (ONOO), and suppression of bacterial mutation, consistent with the earlier observed protection from DNA damage, and prevention of oxidation of lipids and proteins.13

The Mongolian gerbil (Meriones unguiculatus) provides a useful animal model of H. pylori-induced chronic active gastritis that allows investigation of morbidity-related pathological epithelial alterations in gastric mucosa, and their development into intestinal metaplasia and gastric neoplasia.14 The purpose of our study was to evaluate the effectiveness of canolol for inhibition of H. pylori-infected chronic gastritis and gastric carcinogenesis. An investigation of effects on induction of cytokines in mouse peritoneal macrophages in vitro was included.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals

Canolol, 4-vinyl-2,6-dimethoxyphenol (molecular weight, 180) (Fig. 1), is a novel and potent antioxidant, contained in crude canola oil. In our study, it was synthesized to at least 95% purity (confirmed by nuclear magnetic resonance) and mixed with 2,6 di-tert-butyl-4-methylphenol (butylhydroxytoluene, BHT, Sigma Chemical, St Louis, MO) at final concentration of 300 ppm at Junsei Chemical, Tokyo, Japan. The preparation was sealed under helium or nitrogen, and also kept as an ethanol stock solution in nitrogen at −80°C.

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Figure 1. Structure of 4-vinyl-2,6-dimethoxyphenol (canolol). Molecular weight, 180.

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Diets

AIN93G was purchased from Clea Japan, (Tokyo, Japan). Tert-butylhydroquinone (tBHQ), contained in the standard recipe of AIN93G, was excluded to facilitate analysis of subtle changes because of canolol. Soybean oil was replaced with canola oil containing 443.7 ppm tocopherol (158.7 ppm α-tocopherol, 279.6 ppm γ-tocopherol, 5.4 ppm δ-tocopherol and no β-tocopherol) (Showa Sangyo, Funabashi, Japan). Canolol powder containing 300 ppm BHT was dissolved in ethanol and mixed with the modified AIN93G described earlier to give a final concentration of 0.1% canolol and 0.3 ppm BHT. Since no information was available regarding in vivo administration of canolol, previous literature for other antioxidants was referenced for a suitable dose; 0.1–0.5% tocopherol successfully suppressed formation of colon aberrant crypt foci15 and mammary adenocarcinomas.16 Since canolol was revealed to be a more potent oxidative radical scavenger than tocopherol,13 the lowest concentration (0.1%) was applied here. For controls, 0.3 ppm BHT alone or no antioxidant supplement in the basal diet were used. The diets sealed under vacuum were stored in a freezer at −30°C and thawed for use everyday. Leftovers were measured on the next day and the new diet supplied given again to prevent excessive oxidation.

Carcinogen

N-methyl-N-nitrosourea (MNU) (Sigma Chemical, St Louis, MO) was dissolved in distilled water at the concentration of 10 ppm and administered via light-shielded bottles in drinking water ad libitum. MNU solutions were freshly prepared 3 times per week.

Inoculation of H. pylori

ATCC43504 (American Type Culture Collection, Manassas, VA) was grown in Brucella broth (Becton Dickinson, Cockeysville, MD), containing 7% (v/v) heat-inactivated fetal bovine serum, at 37°C under microaerophilic conditions, at high humidity for 24 hr. After 24 hr fasting, gerbils were inoculated via an oral catheter with 1.0 ml aliquots of H. pylori culture containing 1.0 × 108 colony-forming units/ml of the organisms. Four hours later, the animals were again allowed free access to food.

Animals and experimental protocol

One hundred seventy-eight specific-pathogen-free male Mongolian gerbils (MGS/Sea; Seac Yoshitomi, Fukuoka, Japan), 6 weeks old, were used in this study. They were housed in an air-conditioned biohazard room designed for experimentally infected animals, with a 12-hr light/12-hr dark cycle and were allowed free access to food. The experimental design is illustrated in Figure 2. The animals were allocated to Experiments I or II.

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Figure 2. Experimental design. Animals, 6-week-old male Mongolian gerbils; ▾, H. pylori inoculation (i.g.); ▿, Broth only, control. ▪, MNU in drinking water at the concentration of 10 ppm. equation imagecnt; canolol/0.5 ppm 2,6 Di-tert-butyl-4-methylphenol (BHT)/AIN93G diet; equation image, 0.5 ppm BHT/ AIN93G diet, control; equation image, AIN93G diet, control.

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In Experiment I, 58 gerbils were divided into 6 groups (A–F): [Group A, n = 10]: H. pylori-infected, canolol + BHT-treated animals; [Group B, n = 10]: H. pylori-infected, BHT-treated animals, no canolol; [Group C, n = 10]: H. pylori-infected, untreated animals; [Group D, n = 10]: Broth-inoculated (no H. pylori), canolol + BHT-treated animals; [Group E, n = 10]: Broth-inoculated, only BHT-treated animals; and [Group F, n = 8]: Broth-inoculated,untreated animals. Animals were killed at 18 weeks of age for midterm examination.

In Experiment II, 120 gerbils were divided into 4 groups (G to J). Two weeks after inoculation of H. pylori, Groups G, H and I were administrated with MNU for 20 weeks. Group J was given broth and autoclaved distilled water as control. Groups G, H, I and J were given, (i) canolol + BHT, (ii) BHT, (iii) control diet and (iv) canolol + BHT diet, respectively, from the 8th experimental week to the end of the experimental period. BrdU at a dose of 100 mg/kg was injected intraperitoneally 60 min before the sacrifice at experimental week 52. The study was approved by the Animal Care Committee of Aichi Cancer Center Research Institute.

Bacterial cultures

To assess bacterial colonization in the gastric mucosa, half of each glandular stomach from gerbils in Experiment I was homogenized with 1.0 ml of phosphate buffered saline (PBS) for culture of H. pylori. One hundred-microliter aliquots were inoculated onto H. pylori agar plates (Nissui Pharmaceutical, Tokyo, Japan), which were then incubated at 37°C under microaerophilic conditions. The numbers of H. pylori colonies were counted after 5–7 days.

Histopathology and immunohistochemistry

For histological and immunohistochemical examination, the stomachs were fixed in 10% neutral buffered formalin for 24 hr and embedded in paraffin. Serial paraffin sections (4-μm thick) were prepared and stained with hematoxylin and eosin (H&E) for morphological observation, and immunohistochemistry for iNOS and COX-2. Mucosal inflammation in the gastric mucosa was analyzed on H&E-stained sections. The inflammatory responses of glandular mucosa were graded according to the following morphological criteria: Grade 0 (normal), Grade 1 (mild), Grade 2 (moderate) and Grade 3 (marked) (Supplementary Table I). Tissue sections were immunostained for BrdU labeling with a mouse monoclonal anti-BrdU antibody (1:50, DAKO) as described previously.17 Labeling indices (LI) were calculated as the percentages of BrdU-positive epithelial cells within glands. For this purpose 10 different arbitrarily selected points in the antrum and corpus mucosa were selected for quantitation. Immunohistochemical analyses of COX-2 and iNOS were carried out as previously described,18 using an anti-COX-2 mouse monoclonal antibody (diluted 1:200; BD Biosciences, San Jose, CA) and an anti-iNOS mouse polyclonal antibody (diluted 1:500, EMD Biosciences, San Diego, CA). To quantitate the degree of staining, a grading system was employed with the following criteria: Grade 0, no immunoreactivity; Grades 1–3, increasing degrees of intermediate immunoreactivity and Grade 4, extensive immunoreactivity.19

Table I. PCR Primers used for Lightcycler Analysis
GeneSequenceProduct size (bp)
  1. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; F, forward; R, reverse.

GAPDHF: 5′-AACGGCACAGTCAAGGCTGAGAACG-3′118
R: 5′-CAACATACTCGGCACCGGCATCG-3′
 IL-1βF: 5′-TGACTTCACCTTGGAATCCGTCTCT-3′91
R: 5′-GGCAACAAGGGAGCTCCATCAC-3′
 TNF-αF: 5′-GCTGCCCCCACCTCGTGCTC-3′89
R: 5′-CTTGATGGCAGACAGGAGGCTGACC-3′
 COX-2F: 5′-GCCGTCGAGTTGAAAGCCCTCTACA-3′97
R: 5′-CCCCGAAGATGGCGTCTGGAC-3′
 iNOSF: 5′-GCATGACCTTGGTGTTTGGGTGCC-3′110
R: 5′-GCAGCCTGTGTGAACCTGGTGAAGC-3′

Effects of canolol on production of nitric oxide (NO), interleukin-12 (IL-12) and tumor necrosis factor-α (TNF-α) in peritoneal macrophages from mice

Mouse peritoneal macrophages were obtained after intraperitoneal injection of 3 ml of 10% proteosepeptone and Griess assays for nitrate/nitrite measurement were conducted to assess the production of NO. Amounts of IL-12 and TNF-α were measured with enzyme-linked immunosorbent assay (ELISA). Macrophages were incubated with 0–300 μM canolol and 10% FBS at 37°C, then 1.0 μg/ml lipopolysaccharide (LPS) or 1.0 μg/ml LPS plus 0.2 μg/ml interferon-γ (IFN-γ) were added. After incubation for 20 hr, NO, IL-12 and TNF-α production from macrophages was measured.

Analysis of mRNA expression of cytokines by real time quantitative PCR

Total RNA was extracted from the antrum and corpus in the glandular stomach (Experiment I) or in the border region between the 2 regions (Experiment II), using a RNA extraction kit (Isogen, Nippon Gene, Tokyo, Japan). After DNase treatment, first strand cDNAs were synthesized using the Thermoscript RT-PCR System (Invitrogen, Carlsbad, CA) according to the manufacturers' instructions. Quantitative PCR of IL-1β, TNF-α, COX-2 and iNOS, was performed with the LightCycler system (Roche Diagnostics, Mannheim, Germany), using gerbil-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as an internal control. The PCR was performed basically as described using a QuantiTect SYBR Green PCR (QIAGEN) kit with optimal Mg2+ concentration at 2.5 mM. The 5′- and 3′-primer sequences are listed in Table I. Specificity of the PCR reaction was confirmed using the melting program provided with the LightCycler software. To further confirm that there was no obvious primer dimer formation or amplification of any extra bands, the samples were electrophoresed in 3% agarose gels and visualized with ethidium bromide after the LightCycler reaction. Quantification was performed as earlier established using an internal control without any necessity for external standards. The levels of cytokine mRNAs were expressed relative to 1.0 in the control groups (Groups F and J).20

Elevation of 8-OHdG, anti-H. pylori IgG and gastrin in H. pylori infected gerbil plasma

Before the removal of stomachs, blood samples were collected from the inferior vena cava after laparotomy. Sera were separated from blood and their anti-H. pylori IgG antibody titers were measured with an ELISA (GAP-IgG; Biomerica, Newport Beach, CA) and values expressed as an arbitrary index (AI). AI values of more than 1.5 indicated H. pylori infection. Gastrin levels were measured using a radioimmunoassay kit (SRL, Tokyo, Japan). Serum samples were also centrifuged (4°C, 10,000g for 30 min) through centrifugal filter devices (Microcon YM-10, Millipore, Bedford, MA) and measured for 8-OHdG levels (ELISA; high sensitive 8-OHdG check; Japan Institute for Control of Aging, Shizuoka, Japan).21

Statistical analyses

Quantitative values were expressed as means ± SD, and differences between means were evaluated by the Bonferroni multiple-comparison test. p values of less than 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Canolol intake and bacterial colonization

The survival rates of all groups were >95%, with no differences among groups. In the shorter term experiment (Experiment I), total canolol intakes in Groups A and D were 0.45 ± 0.01 and 0.47 ± 0.01 (g/gerbil), respectively. There were no significant differences between Groups A and D. At 12 weeks postinfection, the numbers of H. pylori colonies were 3.17 ± 1.51, 3.52 ± 0.67 and 3.59 ± 0.85 (×104 colony/half stomach) in Groups A, B and C, respectively. No significant inhibitory effect of canolol against bacterial growth was detected (p = 0.64). In the longer term experiment (Experiment II), total canolol intakes in Groups G and J were 1.84 ± 0.02 and 1.84 ± 0.01 (g/gerbil), respectively, again with no significance. There was also no significant variation in body weights (Supplementary Fig. 1) among long-term experiment groups G to J, confirming no apparent toxicity of canolol.

Effects of canolol against H. pylori-induced gastritis and cell proliferation

All gastric mucosal specimens from uninfected gerbils had normal histomorphology. The histological findings for gastric mucosal specimens in H. pylori-infected gerbils are shown in Table II. At 12 weeks (Experiment I), neutrophils and lymphoplasmocytic cell infiltration in the antral mucosa of the canolol-treated group (Group A) were significantly suppressed, compared to the H. pylori-infected control groups (Groups B and C) (Table II; Figs. 3a and 3b). Both antral and corpus BrdU labeling indices in the canolol-treated gerbils (Group A) were significantly reduced as compared to values for control groups B and C (p < 0.05) (Table II). The BrdU LIs in Group A were reduced to 62% in the antrum and 71% in the corpus of the Group B values. During the 52 weeks (Experiment II) there was a change over time in topography of the gastritis, with a shift from predominantly antral gastritis to pangastritis in H. pylori-infected gerbils. Infiltration of inflammatory cells, hyperplasia and intestinal metaplasia lesions of gastric mucosa were markedly lower in the canolol-treated group (Group G) than in the H. pylori-infected control groups (Groups H and I). BrdU LIs in the canolol-treated gerbils (Group G) were again significantly lower both in antrum and corpus than in the control groups H and I, values being decreased to 56 and 64% of the Group H levels (p < 0.05) (Table II).

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Figure 3. Histopathological findings and histology of adenocarcinomas in the gastric mucosa. (a) Mild gastritis in a Group A gerbil at 12 weeks postinfection with canolol treatment. (H&E; ×25); (b) Marked infiltration of inflammatory cells and hyperplasia is seen in a Group B gerbil with control diet (H&E; ×25); (c) Signet-ring cell carcinoma at 52 weeks in a Group H gerbil (H&E; ×200). (d) Well differentiated adenocarcinoma in a glandular stomach at 52 weeks in a Group I gerbil (H&E; ×80).

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Table II. Histopathological Responses in Gerbils
ExperimentsGroupsNo.TreatmentsAntrum
Infiltration of neutrophilsInfiltration of mononuclear cellsHyperplasiaIntestinal metaplasiaBrdU labeling index (%)Score of COX-2 immunohistochemistryScore of iNOS immunohistochemistry
  • Values for results are expressed as means ± SD.

  • 1

    p < 0.01 vs. Groups B and C.–

  • 2

    p < 0.05 vs. Groups B and C.–

  • 3

    p < 0.01 vs. Groups H and I.–

  • 4

    p < 0.05 vs. Groups H and I.

Experiment IA10Hp -> Canolol + BHT1.2 ± 0.412.2 ± 0.411.2 ± 0.610.0 ± 0.012.5 ± 2.510.9 ± 0.210.9 ± 0.11
B10Hp -> BHT2.9 ± 0.33.0 ± 0.12.2 ± 0.30.0 ± 0.020.3 ± 3.72.2 ± 0.21.7 ± 0.2
C10Hp3.0 ± 0.13.0 ± 0.12.3 ± 0.40.0 ± 0.020.9 ± 3.32.3 ± 0.31.5 ± 0.3
D10Broth -> Canolol + BHT0.0 ± 0.00.2 ± 0.10.0 ± 0.00.0 ± 0.05.2 ± 0.80.2 ± 0.10.2 ± 0.2
E10Broth -> BHT0.0 ± 0.00.3 ± 0.10.0 ± 0.00.0 ± 0.05.1 ± 0.80.3 ± 0.10.3 ± 0.1
F8Broth0.0 ± 0.00.3 ± 0.10.0 ± 0.00.0 ± 0.04.2 ± 1.10.3 ± 0.10.2 ± 0.1
 Experiment IIG40Hp + MNU -> Canolol + BHT1.9 ± 0.432.3 ± 0.431.9 ± 0.430.9 ± 0.5310.4 ± 2.331.0 ± 0.431.2 ± 0.53
H33Hp + MNU -> BHT2.6 ± 0.33.0 ± 0.02.5 ± 0.31.7 ± 0.418.5 ± 5.41.7 ± 0.52.1 ± 0.3
I36Hp + MNU2.7 ± 0.23.0 ± 0.02.6 ± 0.41.8 ± 0.420.6 ± 4.61.8 ± 0.51.7 ± 0.5
 J5Broth -> Canolol + BHT0.0 ± 0.00.3 ± 0.10.0 ± 0.00.0 ± 0.04.6 ± 0.60.0 ± 0.00.2 ± 0.1
    Corpus
ExperimentsGroupsNo.TreatmentsInfiltration of neutrophilsInfiltration of mononuclear cellsHyperplasiaIntestinal metaplasiaBrdU labeling index (%)Score of COX-2 immunohistochemistryScore of iNOS immunohistochemistry
Experiment IA10Hp -> Canolol + BHT0.9 ± 0.210.9 ± 0.210.7 ± 0.210.0 ± 0.06.8 ± 1.720.6 ± 0.220.5 ± 0.12
B10Hp -> BHT1.5 ± 0.11.3 ± 0.11.2 ± 0.10.0 ± 0.09.6 ± 2.61.0 ± 0.20.8 ± 0.2
C10Hp1.5 ± 0.21.5 ± 0.21.2 ± 0.10.0 ± 0.010.4 ± 2.71.1 ± 0.10.8 ± 0.2
D10Broth -> Canolol + BHT0.0 ± 0.00.3 ± 0.10.0 ± 0.00.0 ± 0.03.1 ± 0.60.2 ± 0.10.2 ± 0.1
E10Broth -> BHT0.0 ± 0.00.2 ± 0.10.0 ± 0.00.0 ± 0.03.2 ± 0.40.2 ± 0.10.2 ± 0.0
F8Broth0.0 ± 0.00.2 ± 0.10.0 ± 0.00.0 ± 0.02.9 ± 0.50.3 ± 0.10.3 ± 0.1
 Experiment IIG40Hp + MNU -> Canolol + BHT2.2 ± 0.532.5 ± 0.342.1 ± 0.540.8 ± 0.337.9 ± 2.430.6 ± 0.331.0 ± 0.43
H33Hp + MNU -> BHT2.8 ± 0.33.0 ± 0.12.6 ± 0.31.6 ± 0.512.3 ± 2.91.2 ± 0.41.6 ± 0.5
I36Hp + MNU2.7 ± 0.33.0 ± 0.22.8 ± 0.31.7 ± 0.313.8 ± 1.91.3 ± 0.41.4 ± 0.3
J5Broth -> Canolol + BHT0.0 ± 0.00.1 ± 0.10.0 ± 0.00.0 ± 0.04.1 ± 0.70.0 ± 0.00.3 ± 0.2

Immunohistochemistry of COX-2 and iNOS

Immunoreactivity against COX-2 and iNOS was evident in all H. pylori-infected gerbils. However, scores in the canolol-treated groups were significantly lower than in the canolol-untreated control groups (Fig. 4 and Table II).

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Figure 4. Immunohistochemistry of gastric lesions. (a and b) COX-2. Original magnification, ×100. (c and d) iNOS. Original magnification, ×100. Note that the intensity of COX-2 and iNOS immunoreactivity in the canolol-treated gastric mucosa (a and c), is weaker than that in the control infection groups (b and d). (e and f) BrdU. BrdU positive cells are distributed more broadly in the control group (f) than the canolol-treated group (e). Original magnification, ×50.

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Canolol suppression of gastric carcinogenesis

In Experiment II, the incidence of glandular stomach tumors overall was significantly lower in Group G (H. pylori + MNU + canolol + BHT) compared to Groups H (H. pylori + MNU + BHT) [15.0% (6/40) vs. 39.4% (13/33), p = 0.031] and I (H. pylori + MNU) [15.0% (6/40) vs. 41.7% (15/36), p = 0.011] at 52 weeks postinfection (Table III; Figs. 3c and 3d). There was no difference in the incidence of gastric adenocarcinomas between Groups H and I [39.4% (13/33) vs. 41.7% (15/36), p = 1.00]. In the control group J and Experiment I, no tumors developed in the glandular stomach.

Table III. Incidence of Gastric Carcinomas in Gerbils
ExperimentsGroupsNo.TreatmentsCarcinoma
Dif.Undif.Incidence (%)
  • Dif., differentiated adenocarcinoma; Undif., undifferentiated adenocarcinoma. Hp, H.pylori (i.g.).  

  • 1

    p = 0.031 to Group H and p = 0.011 to Group I with Fisher's exact test.

Experiment IIG40Hp + MNU->Canolol + BHT516/40 (15.0)1
H33Hp + MNU->BHT11213/33 (39.4)
I36Hp + MNU15015/36 (41.7)
J5Broth->Canolol + BHT000/5 (0.0)

Suppression of NO and inflammatory cytokines by canolol in mouse peritoneal macrophages in vitro

LPS and IFN-γ induction of NO, IL-12 and TNF-α was significantly inhibited by 50 μM canolol or above in vitro (Figs. 5a5c).

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Figure 5. Suppression of NO and inflammatory cytokine induction in mouse peritoneal macrophages in vitro by canolol. The concentrations of NO (a), IL-12 (b) and TNF-α (c), were significantly reduced by 12.5 μM canolol (means ± SD) (n = 3). *p < 0.05, **p < 0.01 vs. LPS plus IFN-γ.

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Oral administration of canolol and mRNA expression of IL-1β, TNF-α, COX-2 and iNOS

Gastric IL-1β, TNF-α, COX-2 and iNOS were found to be expressed at very low levels in the uninfected control gerbils. However, in the H. pylori-infected groups, the levels of these cytokines and enzymes were markedly elevated in the antrum and corpus already 12 weeks after infection (Experiment I, Fig. 6). Relative expression of IL-1β (Fig. 6a) in Groups B and C was upregulated 16 ± 2 and 16 ± 4 times, respectively, compared to the uninfected group F (control, its value set at 1.0 ± 0.2). Canolol treatment at 0.1% in the diet (Group A) significantly attenuated the increase of mRNA expression to 6.2 ± 1.1 times in the antrum (p < 0.05). For the corpus, it was elevated to 8.0 ± 1.3 (Group B) and 7.6 ± 0.7 (Group C) times with H. pylori infection and decreased to 4.3 ± 0.9 times with canolol treatment (Group A) (p < 0.05) compared to Group F (0.86 ± 0.15 times to corpus of Group F). The figures in Groups D, E and F were comparable. Regarding the expression of TNF-α (Fig. 6b), transcriptional upregulation 29 ± 3 and 25 ± 7 fold in H. pylori infected groups B and C, respectively, was alleviated to 4.0 ± 0.6 times in Group A (p < 0.01). Concerning Cox-2 expression (Fig. 6c), the figures were elevated to 5.3 ± 1.5 (p < 0.01) and 4.5 ± 0.5 (p < 0.05) times in Groups B and C, respectively, and decreased to 1.4 ± 0.7 times in Group A. For iNOS (Fig. 6d), the values were 6.1 ± 0.8 (Group B) and 5.4 ± 0.9 (Group C) times and lowered to 3.4 ± 0.4 times (Group A) in the antrum and 3.6 ± 0.4, 3.4 ± 0.6 and 1.8 ± 0.2 times in the corpus, respectively.

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Figure 6. Relative expression levels of IL-1β, TNF-α, COX-2, and iNOS mRNAs in glandular stomachs of gerbils at 12 weeks postinfection. (a) IL-1β; (b) TNF-α; (c) COX-2; (d) iNOS. Values are arbitrary unit values (mean ± SE) relative to 1.0 for controls. Note decrease in Group A (canolol group) as compared to Groups B and C (controls), especially in the antrum. equation image, Antrum; equation image, corpus. *p < 0.05 and **p < 0.01.

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In Experiment II (Fig. 7), transcription of the inflammatory cytokines reached much higher levels than with the shorter experimental period. IL-1β mRNA was strongly upregulated 116 ± 17 (Group H) and 119 ± 23 (Group I) fold with long term H. pylori infection and this was drastically attenuated to 25 ± 6 times with canolol treatment (Group G, p < 0.01 vs. Groups H and I). TNF-α transcription also increased 93 ± 17 and 101 ± 23 times in Groups H and I and reduced to 13 ± 5 times in Group G (p < 0.01). Regarding Cox-2, the figures of 37 ± 15 and 34 ± 10 times (Groups H and I) were decreased to 6.3 ± 3.2 times (Group G, p < 0.01). Finally, the values of iNOS (28 ± 8 and 30 ± 6 times in Groups H and I, p < 0.05 and p < 0.01, respectively) were again lowered to 15 ± 5 times (Group G).

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Figure 7. Relative expression levels of IL-1β, TNF-α, COX-2 and iNOS mRNAs in glandular stomachs of gerbils at 52 weeks postinfection. (a) IL-1β; (b) TNF-α; (c) COX-2; (d) iNOS. Values are arbitrary unit values (mean ± SE) relative to 1.0 for controls. Note decrease in Group G as compared to Groups H and I. equation image, glandular stomach mucosa at the border between the antrum and corpus. *p < 0.05 and **p < 0.01.

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Effects of canolol on serum 8-OHdG, anti-H. pylori antibodies and gastrin levels

Infection with H. pylori remarkably elevated the serum level of 8-OHdG and anti-H. pylori IgG titers in both Experiments I and II. Significant reduction was noted with canolol treatment (Table IV). After H. pylori infection, serum gastrin levels in Experiment II were elevated at 52 weeks, and this increase was alleviated in the canolol-treated group G (Table IV).

Table IV. Serum 8-OHdG, Anti-H.pylori IgG Titers and Gastrin Levels
ExperimentsGroupsNo.Treatments8-OHdG (ng/ml)Antibody titers (A.I.)Gastrin (pg/ml)
  • 8-OHdG, 8-hydroxy-2′-deoxyguanosine. ND, not determined. Values for results are expressed as means ± SD.

  • 1

    p < 0.05 vs. Groups B and C.

  • 2

    p < 0.01 vs. Groups B and C.

  • 3

    p < 0.01 vs. Groups H and I.

  • 4

    p < 0.05 vs. Groups H and I.

  • 5

    p < 0.05 vs. Groups H and I.

Experiment IA10Hp -> Canolol + BHT0.33 ± 0.05119.6 ± 5.52ND
B10Hp -> BHT0.48 ± 0.1229.8 ± 7.6ND
C10Hp0.51 ± 0.1930.5 ± 8.2ND
D10Broth -> Canolol + BHT0.30 ± 0.051.0 ± 0.4ND
E10Broth -> BHT0.27 ± 0.051.4 ± 0.5ND
F8Broth0.26 ± 0.071.1 ± 0.5ND
 Experiment IIG40Hp + MNU -> Canolol + BHT0.41 ± 0.043186.4 ± 74.24634.0 ± 160.75
H33Hp + MNU -> BHT0.57 ± 0.07249.5 ± 98.5780.7 ± 216.2
I36Hp + MNU0.63 ± 0.12257.9 ± 95.1764.1 ± 195.7
J5Broth -> Canolol + BHT0.29 ± 0.091.1 ± 0.3202.2 ± 54.4

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Triple therapy consisting with a proton pump inhibitor and 2 antimicrobial agents, amoxicillin and clarithromycin, is usually recommended as the general therapy for H. pylori eradication in Japan.22 However, frequent emergence of resistant strains to these antimicrobial agents, and persistence of gastric inflammation even after the eradication of H. pylori, has been observed by physicians. Therefore we need to find means to attenuate gastric inflammation and provide cytoprotection against H. pylori-induced cytotoxicity.23

Our study showed H. pylori-associated chronic active gastritis and gastric carcinogenesis to be effectively suppressed by oral administration of canolol at 0.1% in the diet. In addition, iNOS, COX-2, and inflammatory cytokine IL-1β, IL-12 and TNF-α mRNA expression levels were substantially decreased after canolol administration in vivo and in vitro. It has been reported that a predominantly H. pylori-specific Th1 response, characterized by induction of high level of TNF-α, IL-1β, and IFN-γ is associated with H. pylori-infected gastritis.24, 25 COX-2 and iNOS are well known to play important roles in gastric cancer growth and progression. These results indicate that canolol inhibits the mRNA expression of COX-2, upregulated by H. pylori-infection, and might reduce release of prostaglandin E2 from the gastric mucosa.26 Canolol also suppressed iNOS activity and presumably the NO endogenously produced by this family of enzymes. It is interesting to note that the numbers of H. pylori colonies in the glandular stomach at 12 weeks postinfection was not significantly reduced. Thus, suppression of IL-1β, TNF-α, COX-2 and iNOS, activated by H. pylori-infection, appears critical for the inhibition of gastric carcinogenesis. Crabtree et al.27 showed an increase in the production of TNF-α in antral biopsy specimens from patients with H. pylori gastritis coinciding with neutrophil infiltration. Similarly, Harris et al.28 described the number of mRNA molecules for IL-6 to be elevated to a greater extent in persistently infected rhesus monkeys (6 years) compared to the early phase (7 weeks after infection), whereas expression of IL-1β and TNF-α declined. However, gastric biopsies from persistently infected animals only showed weak gastritis. Yamaoka et al.25 have reported natural history of H. pylori (ATCC43504) induced gastritis and associated gastric mucosal cytokine expression in Mongolian gerbils. In their results, polymorphonuclear and mononuclear cell infiltration was apparent relatively early (8 and 4 weeks after inoculation, respectively) and declined thereafter. Levels of cytokines, including IL-1β, INF-γ, IL-4, IL-6 and IL-10, appeared to be mostly parallel; the values for IFN-γ correlated particularly well with numbers of both polymorphonuclear and mononuclear cells. However, in our experiment, inflammation induced cytokine overexpression was greater in the long-term experiment compared to the short one (Fig. 7vs. Fig. 6). In contrast to the data by Yamaoka et al.,25 infiltration of the inflammatory cells progressively increased. Thus, in terms of the correlation of inflammatory cell infiltrate and cytokine expression level, data in our experiment and their results do not appear to be incompatible. H. pylori inoculated in gerbils might have been partially eradicated or their virulence could have become attenuated in their system.

Of note, at 12 and 52 weeks postinfection, BrdU-labeled cells in gastric mucosa decreased almost 50–70% in canolol-treated gerbils compared to those in H. pylori-infected control groups. Gonzalez et al. found a similar reduction of proliferating cells in ultraviolet B (UVB) exposed mouse epidermis with oral administration of antioxidants like lutein + zexanthin.29 Kim et al. also observed lowering of BrdU LIs with carotenoids (lycopene, fucoxanthin and lutein) and curcumin and its derivative (tetrahydrocurcumin) in 1,2-dimethylhydrazine treated mouse colonic crypts, along with reduced aberrant crypt formation.30 At the molecular level, reactive oxygen intermediates may alter the expression and function of Cox-2 and iNOS, which may influence the expression of proteins involved in regulation of cell cycle progression.31 Similarly, anti-inflammatory and antioxidant agents could protect against such effects and also act on the expression and function of several cell cycle regulating proteins.32

Furthermore, the remarkable elevation of serum 8-OHdG by H. pylori-infection was alleviated by canolol. 8-OHdG has been proposed as a key biomarker of oxidative DNA damage relevant to carcinogenesis,33 because of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anions (O2), singlet oxygen and hydroxyl radicals (OH) as well as reactive nitrogen species (RNS) including ONOO, which is known to cleave DNA and also nitrate guanine to generate 8-nitroguanine. Mutation of Salmonella sp. (TA98) by ONOO was earlier found to be effectively suppressed by canolol,13 one of the most potent anti-ROO antioxidants.13, 34, 35 Therefore, one of the underlying mechanisms is reduction of free radical scavenging oxidative damage.36 In an in vitro system we could also show that canolol suppresses inflammation mediators (Fig. 5).

In conclusion, oral administration of canolol significantly reduced anti-H. pylori IgG antibody titers and gastrin levels in serum, without apparently suppressing H. pylori colonization. A lack of any direct correlation between anti-H. pylori IgG antibody titers and number of colonies was also reported by Murakami et al.37 Canola oil is a traditional cooking oil in many countries. The canolol concentration in crude canola oil is estimated to be ∼220–1,200 ppm, which could provide doses similar to that used in our study. It should be noted, however, that the concentration in refined canola oil is significantly lower12 so that suggestions to the edible oil industry for alternative methods of refining might be warranted. Alternatively, synthesized or extracted canolol could be added to the refined oil and used as table oil or taken as a supplement. Taken together, these findings indicate that the antioxidative compound, canolol, can prevent H. pylori-induced gastritis and carcinogenesis in a gerbil model. Therefore, this dietary factor may have a potential role in controlling H. pylori-associated gastroduodenal diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Ayako Kanazawa (Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan) for valuable information. X. C. was a recipient of a postdoctoral fellowship from the Japan Society for the Promotion of Science during the performance of this research.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Parsonnet J,Friedman GD,Vandersteen DP,Chang Y,Vogelman JH,Orentreich N,Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991; 325: 112731.
  • 2
    Uemura N,Okamoto S,Yamamoto S,Matsumura N,Yamaguchi S,Yamakido M,Taniyama K,Sasaki N,Schlemper RJ. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 7849.
  • 3
    Danese S,Papa A,Gasbarrini A,Ricci R,Maggiano N. Helicobacter pylori eradication down-regulates matrix metalloproteinase-9 expression in chronic gastritis and gastric ulcer. Gastroenterology 2004; 126: 36971.
  • 4
    Wong BC,Lam SK,Wong WM,Chen JS,Zheng TT,Feng RE,Lai KC,Hu WH,Yuen ST,Leung SY,Fong DY,Ho J, et al. Helicobacter pylori eradication to prevent gastric cancer in a high-risk region of China: a randomized controlled trial. JAMA 2004; 291: 18794.
  • 5
    Graham DY. Antibiotic resistance in Helicobacter pylori: implications for therapy. Gastroenterology 1998; 115: 12727.
  • 6
    Matsubara S,Shibata H,Ishikawa F,Yokokura T,Takahashi M,Sugimura T,Wakabayashi K. Suppression of Helicobacter pylori-induced gastritis by green tea extract in Mongolian gerbils. Biochem Biophys Res Commun 2003; 310: 71519.
  • 7
    Iimuro M,Shibata H,Kawamori T,Matsumoto T,Arakawa T,Sugimura T,Wakabayashi K. Suppressive effects of garlic extract on Helicobacter pylori-induced gastritis in Mongolian gerbils. Cancer Lett 2002; 187: 618.
  • 8
    Otsuka T,Tsukamoto T,Tanaka H,Inada K,Utsunomiya H,Mizoshita T,Kumagai T,Katsuyama T,Miki K,Tatematsu M. Suppressive effects of fruit-juice concentrate of Prunus mume Sieb. et Zucc. (Japanese apricot, Ume) on Helicobacter pylori-induced glandular stomach lesions in Mongolian gerbils. Asian Pac J Cancer Prev 2005; 6: 33741.
  • 9
    Kim H,Seo JY,Kim KH. Effect of mannitol on Helicobacter pylori-induced cyclooxygenase-2 expression in gastric epithelial AGS cells. Pharmacology 2002; 66: 1829.
  • 10
    Correa P,Fontham ET,Bravo JC,Bravo LE,Ruiz B,Zarama G,Realpe JL,Malcom GT,Li D,Johnson WD,Mera R. Chemoprevention of gastric dysplasia: randomized trial of antioxidant supplements and anti-Helicobacter pylori therapy. J Natl Cancer Inst 2000; 92: 18818.
  • 11
    Sun YQ,Girgensone I,Leanderson P,Petersson F,Borch K. Effects of antioxidant vitamin supplements on Helicobacter pylori-induced gastritis in Mongolian gerbils. Helicobacter 2005; 10: 3342.
  • 12
    Wakamatsu D,Morimura S,Sawa T,Kida K,Nakai C,Maeda H. Isolation, identification, and structure of a potent alkyl-peroxyl radical scavenger in crude canola oil, canolol. Biosci Biotechnol Biochem 2005; 69: 156874.
  • 13
    Kuwahara H,Kanazawa A,Wakamatu D,Morimura S,Kida K,Akaike T,Maeda H. Antioxidative and antimutagenic activities of 4-vinyl-2,6-dimethoxyphenol (canolol) isolated from canola oil. J Agric Food Chem 2004; 52: 43807.
  • 14
    Tatematsu M,Tsukamoto T,Inada K. Stem cells and gastric cancer—role of gastric and intestinal mixed intestinal metaplasia. Cancer Sci 2003; 94: 13541.
  • 15
    Newmark HL,Huang MT,Reddy BS. Mixed tocopherols inhibit azoxymethane-induced aberrant crypt foci in rats. Nutr Cancer 2006; 56: 825.
  • 16
    Hirose M,Nishikawa A,Shibutani M,Imai T,Shirai T. Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environ Mol Mutagen 2002; 39: 2718.
  • 17
    Ikeno T,Ota H,Sugiyama A,Ishida K,Katsuyama T,Genta RM,Kawasaki S. Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils. Am J Pathol 1999; 154: 95160.
  • 18
    Tanaka T,Kohno H,Suzuki R,Hata K,Sugie S,Niho N,Sakano K,Takahashi M,Wakabayashi K. Dextran sodium sulfate strongly promotes colorectal carcinogenesis in Apc(Min/+) mice: inflammatory stimuli by dextran sodium sulfate results in development of multiple colonic neoplasms. Int J Cancer 2006; 118: 2534.
  • 19
    Zingarelli B,Szabo C,Salzman AL. Reduced oxidative and nitrosative damage in murine experimental colitis in the absence of inducible nitric oxide synthase. Gut 1999; 45: 199209.
  • 20
    Tsukamoto T,Fukami H,Yamanaka S,Yamaguchi A,Nakanishi H,Sakai H,Aoki I,Tatematsu M. Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase a and b subunit mRNAs, in colon of 1,2-dimethylhydrazine-treated rats. Jpn J Cancer Res 2001; 92: 10918.
  • 21
    Magari H,Shimizu Y,Inada K,Enomoto S,Tomeki T,Yanaoka K,Tamai H,Arii K,Nakata H,Oka M,Utsunomiya H,Tsutsumi Y, et al. Inhibitory effect of etodolac, a selective cyclooxygenase-2 inhibitor, on stomach carcinogenesis in Helicobacter pylori-infected Mongolian gerbils. Biochem Biophys Res Commun 2005; 334: 60612.
  • 22
    Malfertheiner P,Megraud F,O'Morain C,Bell D,Bianchi Porro G,Deltenre M,Forman D,Gasbarrini G,Jaup B,Misiewicz JJ,Pajares J,Quina M, et al. Current European concepts in the management of Helicobacter pylori infection—the Maastricht Consensus Report. The European Helicobacter pylori Study Group (EHPSG). Eur J Gastroenterol Hepatol 1997; 9: 12.
  • 23
    Wang WH,Wong BC,Mukhopadhyay AK,Berg DE,Cho CH,Lai KC,Hu WH,Fung FM,Hui WM,Lam SK. High prevalence of Helicobacter pylori infection with dual resistance to metronidazole and clarithromycin in Hong Kong. Aliment Pharmacol Ther 2000; 14: 90110.
  • 24
    D'Elios MM,Manghetti M,De Carli M,Costa F,Baldari CT,Burroni D,Telford JL,Romagnani S,Del Prete G. T helper 1 effector cells specific for Helicobacter pylori in the gastric antrum of patients with peptic ulcer disease. J Immunol 1997; 158: 9627.
  • 25
    Yamaoka Y,Yamauchi K,Ota H,Sugiyama A,Ishizone S,Graham DY,Maruta F,Murakami M,Katsuyama T. Natural history of gastric mucosal cytokine expression in Helicobacter pylori gastritis in Mongolian gerbils. Infect Immun 2005; 73: 220512.
  • 26
    Romano M,Ricci V,Memoli A,Tuccillo C,Di Popolo A,Sommi P,Acquaviva AM,Del Vecchio Blanco C,Bruni CB,Zarrilli R. Helicobacter pylori up-regulates cyclooxygenase-2 mRNA expression and prostaglandin E2 synthesis in MKN 28 gastric mucosal cells in vitro. J Biol Chem 1998; 273: 285603.
  • 27
    Crabtree JE,Shallcross TM,Heatley RV,Wyatt JI. Mucosal tumour necrosis factor a and interleukin-6 in patients with Helicobacter pylori associated gastritis. Gut 1991; 32: 14737.
  • 28
    Harris PR,Smythies LE,Smith PD,Dubois A. Inflammatory cytokine mRNA expression during early and persistent Helicobacter pylori infection in nonhuman primates. J Infect Dis 2000; 181: 7836.
  • 29
    Gonzalez S,Astner S,An W,Goukassian D,Pathak MA. Dietary lutein/zeaxanthin decreases ultraviolet B-induced epidermal hyperproliferation and acute inflammation in hairless mice. J Invest Dermatol 2003; 121: 399405.
  • 30
    Kim JM,Araki S,Kim DJ,Park CB,Takasuka N,Baba-Toriyama H,Ota T,Nir Z,Khachik F,Shimidzu N,Tanaka Y,Osawa T, et al. Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation. Carcinogenesis 1998; 19: 815.
  • 31
    Shackelford RE,Kaufmann WK,Paules RS. Oxidative stress and cell cycle checkpoint function. Free Radic Biol Med 2000; 28: 1387404.
  • 32
    Baldassarre G,Nicoloso MS,Schiappacassi M,Chimienti E,Belletti B. Linking inflammation to cell cycle progression. Curr Pharm Des 2004; 10: 165366.
  • 33
    Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 1997; 387: 14763.
  • 34
    Akaike T,Ijiri S,Sato K,Katsuki T,Maeda H. Determination of peroxyl radical-scavenging activity in food by using bactericidal action of alkyl peroxyl radical. J Agric Food Chem 1995; 4: 186470.
  • 35
    Kanazawa A,Sawa T,Akaik T,Maeda H. Formation of abasic sites in DNA by t-butyl peroxyl radicals: implication for potent genotoxicity of lipid peroxyl radicals. Cancer Lett 2000; 156: 515.
  • 36
    Okazaki K,Ishii Y,Kitamura Y,Maruyama S,Umemura T,Miyauchi M,Yamagishi M,Imazawa T,Nishikawa A,Yoshimura Y,Nakazawa H,Hirose M. Dose-dependent promotion of rat forestomach carcinogenesis by combined treatment with sodium nitrite and ascorbic acid after initiation with N-methyl-N′-nitro-N-nitrosoguanidine: possible contribution of nitric oxide-associated oxidative DNA damage. Cancer Sci 2006; 97: 17582.
  • 37
    Murakami M,Ota H,Sugiyama A,Ishizone S,Maruta F,Akita N,Okimura Y,Kumagai T,Jo M,Tokuyama T. Suppressive effect of rice extract on Helicobacter pylori infection in a Mongolian gerbil model. J Gastroenterol 2005; 40: 45966.

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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