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|Sulforaphane effective against H. Pylori|
|Sulforaphane - could fight Leukemia, cancers||
Fortified Natural Organic Flax Hulls
(with 3-day old broccoli sprouts)
contains high levels of natural botanical flax lignans and sulforaphane from broccoli sprouts
Efficacy of Sulforaphane in Eradicating Helicobacter pylori in Human Gastric Xenografts Implanted in
Xavier Haristoy,1 Karine Angioi-Duprez,2 Adrien Duprez,3 and Alain Lozniewski1*
Received November 15, 2002; Revised May 24, 2003; Accepted August 25, 2003.
Sulforaphane, an isothiocyanate abundant in the form of its glucosinolate precursor in broccoli sprouts, has
shown in vitro activity against Helicobacter pylori. We evaluated the effect of sulforaphane in vivo against this bacterium by
using human gastric xenografts in nude mice. H. pylori was completely eradicated in 8 of the 11 sulforaphane-treated
grafts. This result suggests that sulforaphane might be beneficial in the treatment of H. pylori-infected individuals.
The recommended treatment of Helicobacter pylori-associated diseases today includes a combination of two or more antibiotics with an inhibitor
of acid secretion. However, even with these treatments, some bacteria may persist, in a nongrowing, tolerant form or possibly intracellularly
(5, 7, 10). This may lead to eradication failure, which is defined as the persistence of H. pylori at least 1 month after the end of
antimicrobial therapy(9). Moreover, development of resistance of H. pylori strains to one or more of the antibiotics commonly used
has been demonstrated previously(28). Thus, a need for new therapeutic agents that are effective against extracellular and
potentially intracellular forms of H. pylori exists. We recently showed that sulforaphane, an isothiocyanate abundant in the form of its
glucosinolate precursor in broccoli and broccoli sprouts, is a potent bactericidal agent against both extra- and intracellular H. pylori in
vitro(11). Substantial quantities of isothiocyanates (up to 100 mg daily) and even greater quantities of their glucosinolate
precursors are widely consumed by humans(12, 13, 29). They may act locally within the gastrointestinal tract or may distribute
systemically after conversion to their cognate isothiocyanates(12, 15, 34). In this study, we investigated the efficacy of
sulforaphane in vivo against H. pylori by using a recently developed model which uses human gastric xenografts in nude mice(22).
Sulforaphane, i.e., [(-)-1-isothiocyanato-(4R)-(methylsulfinyl)butane] was kindly provided by J. W. Fahey (Johns Hopkins University School
of Medicine, Baltimore, Md.). Stock solutions were prepared in acetonitrile, and further dilutions were made in sterile water. H. pylori
strain 26695, which was previously adapted for use in the gastric xenograft model(23), was used for graft inoculation. The strain
was grown on Columbia agar supplemented with 10% horse blood under microaerobic conditions as previously described(22). For this
isolate, the MIC of sulforaphane was 4 Ág/ml, as determined by using the agar dilution method (pH 7.4) as recommended by the National
Committee for Clinical Laboratory Standard (NCCLS)(30).
Xenografts exhibiting human mature gastric epithelium and acidic secretion were obtained in nude mice as previously described(22)
and with the approval of the French National Consultative Ethical Committee. Bacterial inoculation was performed by using a catheter that was
implanted in the xenograft lumen(22). Two weeks after inoculation, each graft was microsurgically opened. Mucus was sampled for
qualitative culture onto blood agar, and three biopsies were taken from antrum-adjacent sites for quantitative culture and histology. The
levels of colonization and the concentrations of intracellular bacteria were determined by quantitative culture as previously described
(23). For histological studies, specimens were processed by standard methods and stained with hematoxylin-eosin to assess the intensity
of gastritis and with a modified Giemsa stain for detection of H. pylori(22).
The grafted animals were then randomly divided into two groups of 11. In the first group, 0.5 ml of sterile water with 0.5% acetonitrile
containing 7.5 Ámol of sulforaphane (approximately 1.33 mg) was administered via catheter once a day for 5 days. The same solution without
sulforaphane was administered in the same way for 5 days in the other 11 infected grafts (control group). The effect of sulforaphane on H.
pylori eradication was evaluated 4 weeks after treatment per the previous recommendation for human subjects(9). At that time, the
animals were euthanized and the grafts were removed and opened. Three biopsy specimens were then taken from adjacent sites in the antrum for
histological examination and determination of the level of mucosal colonization as well as the concentration of intracellular bacteria
concentrations as described above. Eradication was defined by the absence of H. pylori detection by both histology and culture methods.
Personnel performing this evaluation were masked (blinded) with regard to the treatment status. The MIC of sulforaphane was determined for
all recovered isolates obtained from each culture-positive graft.
Before treatment, all 22 inoculated grafts were equally infected by H. pylori 26695, with no significant differences in numbers of CFU between
the grafts that were destined to receive sulforaphane and the controls (P = 0.34, Mann-Whitney U test) (Table 1). No organism other than H.
pylori was recovered from either mucus or mucosal samples. Viable intracellular bacteria were also isolated in all the grafts and represented
0.04 to 2.5% (mean, 0.9%) of all the viable bacteria detected in the gastric mucosa. At that time, rare limited erythematous areas associated
with hemorrhagic points or ulcerations were observed at the surface of the antrum in all inoculated grafts. Histological examination of
antral biopsies confirmed the presence of H. pylori and showed mild inflammation and mild or moderate activity associated with diffuse
interstitial edema. During the administration of sulforaphane or placebo (diluent vehicle control) and throughout the posttherapy period, no
adverse reaction or change in weight or behavior was observed in the mice. Moreover, we did not observe any macroscopic or histopathological
changes in internal organs at the end of the experiments. One month after the end of treatment, eradication was observed in 8 of the 11
sulforaphane-treated grafts (Table 1). Neither macroscopic abnormalities nor antral gastritis were observed in these culture-negative grafts.
For the control group, there was no significant difference between the mucosal bacterial concentrations found before administration of the
diluent and those found 5 weeks later (P = 0.10, Mann-Whitney U test) (Table 1). In sulforaphane-treated grafts that were not eradicated and
in control grafts, both macroscopic and histological features were similar to those observed before treatment. Intracellular bacteria were
detected in all control grafts (1 to 2.7% of all the viable bacteria detected) and in two of the three sulforaphane-treated grafts that were
not eradicated (0.04 and 0.3% of all the viable bacteria detected). For isolates obtained from control grafts, no change in the MIC of
sulforaphane was observed, whereas a fourfold increase of the MIC (to 16 Ág/ml) was observed for all the isolates obtained from the three
sulforaphane-treated grafts from which H. pylori was not eradicated. Such organisms were not detected after plating of the initial inoculum
onto sulforaphane-containing agar.
A wide variety of plant extracts, i.e., individual plant components such as phytochemicals and phytochemical mixtures, have been shown to be
active in vitro against H. pylori(1, 3, 4, 6, 8, 17-21, 24-26, 32, 33, 35, 36). A few of these phytochemicals have been tested in
vivo(2, 14, 16, 20, 24, 27, 31), but clearance of H. pylori was observed only in two cases: after administration of tea catechins
to H. pylori-infected gerbils(24) and after administration of goshuyu-to, a medicinal preparation which contains various plant
extracts, including ginseng, to humans(16). It is therefore critical to subject all plant compounds that are active against H.
pylori in vitro to in vivo studies to determine their effectiveness in whole-organism systems, since most plant-derived compounds have not
proven efficacious in such testing. Thus, having recently shown that sulforaphane was active against H. pylori in vitro and that it prevented
the formation of benzo[a]pyrene-induced stomach tumors in ICR mice following an estimated daily intake of 7.5 Ámol for 5 weeks(11),
we next evaluated its potency in eradicating H. pylori, using the same dose for 5 days, in human gastric xenografts that were implanted in
nude mice. In the present work, we have shown that sulforaphane has a significant effect against H. pylori in vivo, since we observed an
eradication rate of 73% for the treated group and noted no eradication in the control group. Previous studies have demonstrated that
sulforaphane, which has a concentration-dependent bactericidal activity against H. pylori, may accumulate in H. pylori-infected cells and
reach intracellular levels at least fivefold higher than the administered concentration, suggesting that a reason for its bactericidal activity
against intracellular bacteria may be related to its accumulation in mammalian cells(11). Whether intracellular accumulation of
sulforaphane occurs in vivo is not yet known, but this may be one reason that the effectiveness of sulforaphane was not influenced in our
study by the presence of intracellular bacteria. A failure of complete eradication was observed in only 3 of the 11 sulforaphane-treated
grafts (due to the nature of the experiment, it was impossible to know whether there was a transient effect on H. pylori colonization), and
such failure was always associated with the presence of isolates for which the MIC of sulforaphane was fourfold higher than that during the
initial inoculation of an isolate. This apparent reduction in susceptibility may explain, at least partially, the failure of sulforaphane to
completely eradicate H. pylori in these three xenografts. Other reasons, such as the existence of subtherapeutic antibiotic concentrations or
poor stability of the drug at the site of infection, cannot be excluded.
We showed that H. pylori can be eradicated from human gastric xenografts after short-term administration of sulforaphane at a dose (1.33 mg/day
in each xenograft [volume, ~7 ml]; 0.19 mg/ml) that can be achieved in the human diet (100 mg/day [stomach volume, 0.5 to 1 liter]; 0.1 to 0.2
mg/liter)(12, 13, 29). Thus, the administration of sulforaphane that can be safely delivered in the diet, particularly from
broccoli sprouts, could be beneficial for the treatment of H. pylori-associated gastric diseases. A sulforaphane-enriched diet might also be
of value for prophylaxis against H. pylori infection and should be further evaluated. Xenografts represent the only in vivo model that permits
the study of the effect of a compound on H. pylori interacting with fully differentiated human gastric mucosa. Since this model does not
completely mimic the microenvironment of the infected human stomach, which is subject to the action of food, digestive physiology, and the
potential coexistence of other bacterial species, further studies in humans are necessary to confirm the in vivo activity of sulforaphane
against H. pylori.
We thank Jed W. Fahey, Katherine K. Stephenson, and Paul Talalay (Department of Pharmacology and Molecular Sciences, The Johns Hopkins
University School of Medicine, Baltimore, Md.) for their gift of sulforaphane and for their expert editorial advice.
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Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of
American Society for Microbiology (ASM)
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