APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2005, p. 8558–8564
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 12
Inhibition of Helicobacter pylori and Associated Urease by
Oregano and Cranberry Phytochemical Synergies
Y. T. Lin, Y. I. Kwon, R. G. Labbe, and K. Shetty*
Department of Food Science, Chenoweth Laboratory, University of Massachusetts, Amherst, Massachusetts 01003
Received 8 May 2005/Accepted 13 September 2005
Ulcer-associated dyspepsia is caused by infection with Helicobacter pylori. H. pylori is linked to a majority of
peptic ulcers. Antibiotic treatment does not always inhibit or kill H. pylori with potential for antibiotic
resistance. The objective of this study was to determine the potential for using phenolic phytochemical extracts
to inhibit H. pylori in a laboratory medium. Our approach involved the development of a specific phenolic
profile with optimization of different ratios of extract mixtures from oregano and cranberry. Subsequently,
antimicrobial activity and antimicrobial-linked urease inhibition ability were evaluated. The results indicated
that the antimicrobial activity was greater in extract mixtures than in individual extracts of each species. The
results also indicate that the synergistic contribution of oregano and cranberry phenolics may be more
important for inhibition than any species-specific phenolic concentration. Further, based on plate assay, the
likely mode of action may be through urease inhibition and disruption of energy production by inhibition of
proline dehydrogenase at the plasma membrane.
Dyspepsia and related problems with peptic ulcers linked to
Helicobacter pylori are major problems in many parts of the
world (37). H. pylori is a gram-negative, spiral-shaped bacte-
rium that lives in the stomach and duodenum. By releasing an
enzyme called “urease,” H. pylori is able to survive in the
stomach. Urease converts urea into ammonia, which then
counters the stomach acid. This creates a neutralizing environ-
ment for protecting H. pylori from the acid in the stomach.
Gastric infection with H. pylori may lead to the onset of various
gastric-related diseases (13). Most patients specifically with
duodenal ulcer can be cured by killing H. pylori with antibiotics
and proton pump inhibitors (22, 29). In recent studies, H. pylori
infection was also suspected to be associated with coronary
artery and ischemic heart disease (4, 9, 21, 23). Many antibi-
otic-linked treatments have been recommended for eradica-
tion of H. pylori, but the emergence of antibiotic resistance
makes the treatments more complicated, and the infection is
sustained at higher levels when the drug treatment is stopped
(12, 29). Two common antibiotics used for treatment of H.
pylori infection are metronidazole and clarithromycin. Several
triple- or quadruple-antibiotic therapies with proton pump in-
hibitors have been shown to be effective in eradication of H.
pylori (14), but no single treatment regimen is considered the
final treatment of choice.
Research has indicated that urease of H. pylori is located in
the cytoplasm in freshly prepared cultures and in the outer
membrane in older cultures (15). In addition to pathogenicity
from H. pylori, evidence indicates that ammonia generated by
urease can cause injury to the gastroduodenal mucosa (33, 42).
Specific inhibition of urease activity has been proposed as a
possible strategy to inhibit this microorganism (25). It has been
demonstrated that a urease-negative mutant does not cause
gastritis in nude mice due to difficulty in colonization (40).
These results suggest the important role of urease in bacterial
Many naturally occurring compounds found in dietary and
medicinal plants, herbs, and fruit extracts have been shown to
possess antimicrobial activities (7, 18, 19, 41). Recent research
has indicated that some key phenolic phytochemicals in plant
extracts have antimicrobial properties that inhibit the bacteria
that cause common types of food poisoning, such as the food-
borne pathogens Listeria monocytogenes (20, 30) and Staphy-
lococcus aureus (1). These results also indicated the potential
of using plant extracts as antimicrobial ingredients in food to
inhibit H. pylori (10, 16, 24, 34, 38). Previous research also
indicated that host antioxidant stimulation is related to en-
hanced H. pylori inhibition (2). Therefore, we have proposed to
develop a specific phenolic antioxidant profile to inhibit H.
pylori. Our strategy couples the benefits of antioxidant activity
with specific phenolic profiles to inhibit H. pylori. Further, we
have previously investigated whether botanical phytochemical
mixtures contribute to antioxidant functionality and antimicro-
bial effects through synergy (41). In the present study, we also
made initial investigations into the likely mode of action by
using simple plate assays to evaluate inhibition of urease and
proline dehydrogenase at the plasma membrane level.
MATERIALS AND METHODS
Preparation of plant extracts. Water-soluble oregano (Origanum vulgare) and
cranberry (Vaccinium macrocarpon) extract powders were obtained from Bar-
rington Chemicals, NY, and Decas Cranberry Products, Wareham, MA, respec-
tively. Ten grams of different ratios of oregano/cranberry powder mixture (Table 1)
were added to 90 ml water to make concentrated stock extracts and stored at 4°C.
Different ratios of concentrated extracts were then diluted on a total phenolic
basis and filter sterilized before use. The total phenolic contents in oregano and
cranberry powders were a minimum of 16 mg/g (wt/wt) (dry weight basis) and 5
mg/g (wt/wt), respectively. The major phenolic found in oregano extracts was
rosmarinic acid at a minimum of 3% of total phenolics. Cranberry extracts had
a range of phenolic compounds, such as ellagic acid, p-hydroxybenzoic acid,
cinnamic acid, hydroxycinnamic acid, caffeic acid, chlorogenic acid, ferulic acid,
* Corresponding author. Mailing address: Department of Food Sci-
ence, Chenoweth Laboratory, University of Massachusetts, Amherst,
MA 01003. Phone: (413) 545-1022. Fax: (413) 545-1262. E-mail:
sinapic acid, and p-coumaric acids (43), with each being less than 0.5% of total
Total phenolic assay. The total phenolic content was determined spectropho-
tometrically by the Folin-Ciocalteu assay. This method was originally developed
by Chandler and Dodds (5) and later modified for oregano phenolics (31).
Approximately 50 mg of different ratios of phytochemical powder mixtures were
added into 150- by 15-mm glass tubes. Distilled water (2.5 ml) was added to the
tubes. The samples were homogenized using a Tissue-Tearor (Biospec Products,
Bartleville, OK) and centrifuged at 13,000 ? g for 10 min (25°C). One milliliter
of the supernatant fluid was transferred to a test tube, to which 6 ml of distilled
water and 0.5 ml of Folin-Ciocalteu phenol reagent (Sigma Chemical Co., St.
Louis, MO) were added. After incubation for 5 min (25°C), 1 ml of 5% Na2CO3
was added. The tubes were mixed well and kept in the dark (25°C) for an hour.
The samples were vortexed, and absorbance at 725 nm was measured (Spectronic
Genesys 5; MiltonRoy Company, Rochester, NY). The phenolic content was
then calculated from a gallic acid standard curve.
Agar diffusion assay. H. pylori was cultured using the method of Stevenson et
al. (35). The isolate of H. pylori (strain ATCC 43579, which originated from
human gastric samples) was obtained from the American Type Culture Collec-
tion (Manassas, VA). Standard plating medium were prepared by using 10 g of
special peptone (Oxoid Ltd., Basingstoke, England), 15 g of granulated agar
(Difco Laboratories, Detroit, MI), 5 g of sodium chloride (EM Science,
Gibbstown, NJ), 5 g of yeast extract (Difco), and 5 g of beef extract (Becton
Dickinson and Co., St. Louis, MO) per liter of water. Activity against H. pylori
was tested by the standard agar diffusion method. Broth media were prepared in
the same way without agar. One hundred microliters of stock H. pylori was added
to test tubes containing 10 ml of broth medium. The tubes were incubated at
37°C for 48 h before being used for spread plate assays.
Agar diffusion assay was done aseptically using sterile 1/4-in. (0.635-cm)-
diameter paper disks (Schleicher & Schuell, Inc., Keene, NH). Individual phy-
tochemical extracts and mixtures of extracts were added to paper disks (100 ?l,
containing a total phenolic content of 0.1 mg) by using a micropipette. Phyto-
chemical-saturated disks were placed on surfaces of seeded agar plates. Plates
were incubated at 37°C for 48 h in GasPak jars (BBL Microbiology Systems,
Cockeysville, MD) with BBL CampyPacks (BBL Microbiology Systems,
Cockeysville, MD). The diameter of the inhibition zone surrounding each disk
was measured, and the zone of inhibition was determined. Various combinations
of oregano and cranberry (Table 1) were evaluated for antimicrobial efficacy.
Controls consisted of disks with distilled water only.
A2C/proline assay. The antimicrobial effect of phenolic phytochemicals was
compared to that of azetidine-2-carboxylate (A2C) based on the rationale that
small phenolics in phytochemical profiles could behave like proline analogs and
likely inhibit proline dehydrogenase (32). Further the effects of phenolic phyto-
chemicals or A2C could be overcome by proline if the site of action is proline
H. pylori was cultured using the method of Stevenson et al. (35). Plating
medium were prepared by using standard plating medium as described for the
agar diffusion assay, with some modifications. Proline (Sigma, St. Louis, MO)
was added to the medium to give a final concentration of 1.0 mM. The antimi-
crobial assay was done in the same way as the agar diffusion assay. Individual
phytochemical extracts and mixtures were added to paper disks (100 ?l, contain-
ing 0.1 mg of total phenolics) using a micropipette. A2C was prepared at a
concentration of 0.1 mM and added at 100 ?l to paper disk. Saturated disks were
placed on the surfaces of seeded agar plates. Plates were incubated at 37°C for
48 h in GasPak jars (BBL Microbiology Systems, Cockeysville, MD) with BBL
CampyPacks (BBL Microbiology Systems, Cockeysville, MD). The diameter of
the inhibition zone surrounding each disk was measured, and the zone of inhi-
bition was determined. Each experiment consisted of three replicates with var-
ious phytochemical concentrations. Each experiment was repeated three times.
Controls consisted of disks with distilled water only.
Assay of urease activity in disks. We developed the urease plate assay based
on the rationale that if urease is located in the cytoplasmic membrane or ex-
creted by the bacteria under low-pH conditions, it will convert urea to ammonia
and counter the low pH. When this happens, bromocresol purple will be con-
verted to purple due to the pH increase. If urease activity was inhibited, a yellow
zone would be observed due to low pH. Plating medium was modified from the
standard plating medium described for the agar diffusion assay. Urea (Schwarz/
Mann Biotech, Cleveland, OH) was added to the medium to a final concentra-
tion of 10 mM. Bromocresol purple was added to the medium at 0.01 g per liter.
The final pH of the medium was adjusted to 6.0. Individual phytochemical
extracts and mixtures (pH 7.0) were adding to paper disks (50 ?l, containing 0.05
mg of total phenolics) by using a micropipette. Saturated disks were placed on
the surfaces of seeded agar plates. Plates were incubated at 37°C for 48 h in
GasPak jars (BBL Microbiology Systems, Cockeysville, MD) with BBL Campy-
Packs (BBL Microbiology Systems, Cockeysville, MD). The diameter of the
yellow zone surrounding each disk was measured. Each experiment consisted of
three replicates with various phytochemical concentrations. Each experiment was
repeated three times. Controls consisted of disks with distilled water only.
Assay of urease activity in broth (26). Urease activity of H. pylori was also
determined by measuring the release of ammonia by a modification of the
Berthelot reaction (8). Special peptone broth was made from the plating medium
described for the agar diffusion assay but without agar. The pH of the broth was
adjusted to 6.0 prior to use. Cells were grown in the special peptone broth for
48 h at 37°C (A560of 1.0) and then incubated with oregano, cranberry, and an
extract mixture (25%/75%) at a concentration of 0.05 mg phenolic/ml for 10 min
at 28°C. The incubated cell cultures were centrifuged at 4°C (4,000 ? g, 5 min)
and resuspended in 0.5 volume of ice-cold 0.1 M sodium phosphate buffer (pH
7.3) containing 10 mM EDTA. Cells were disrupted by sonication, and the
supernatant obtained after centrifugation at 4°C (12,000 ? g, 5 min) was used for
the urease assay. The reaction mixture contained 50 mM urea, 100 mM sodium
phosphate buffer (pH 7.3), and an aliquot of the supernatant in a total volume of
1.0 ml. After incubation for 10 min at 37°C, the reaction was terminated by
addition of 2 ml of 0.5% phenol and 0.0025% sodium nitroprusside solution,
after which 2 ml of 0.25% sodium hydroxide and 0.21% sodium hypochlorite
solution were added, the mixture was incubated for 6 min at 55°C for color
development, and the absorbance at 625 nm was determined. Blanks were cells
treated similarly but without phytochemical mixtures. The amount of ammonia
produced was equivalent to the hydrolysis of urea. A high absorption value
indicated high urease activity in the supernatant.
Total protein assay. Cell cultures with phytochemical extracts prepared in the
previous urease assay were then used for total protein assay. The samples were
centrifuged at 4°C (4,000 ? g, 5 min) and resuspended in 0.5 volume of ice-cold
0.1 M sodium phosphate buffer (pH 7.3) containing 10 mM EDTA. Cells were
disrupted by sonication, and the supernatant was obtained after centrifugation at
4°C (12,000 ? g, 5 min). The supernatant was used for the estimation of total
Protein content was measured by the method of Bradford (3). The dye reagent
concentrate (Bio-Rad protein assay kit II; Bio-Rad Laboratory, Hercules, CA)
was diluted 1:4 with distilled water. Five milliliters of diluted dye reagent was
added to 100 ?l of the supernatant. After vortexing and incubating for 5 min, the
absorbance was measured at 595 nm against a 5-ml reagent blank and 100 ?l
buffer by using a UV-visible Genesys spectrophotometer (Milton Roy, Inc.,
Rochester, NY). The urease activity was expressed as the amount of ammonia
produced per unit protein compared to the control without phytochemical ex-
RESULTS AND DISCUSSION
Agar diffusion assay. A level of 0.1 mg phenolic/disk was
the concentration determined from preliminary investiga-
tions to be the effective dose. The results indicated that the
best antimicrobial activity against H. pylori was observed
when the mixture contained 25% (wt/wt) oregano with 75%
(wt/wt) cranberry (Fig. 1). At pH 7.0, disks containing this
extract mixture (0.1 mg total phenolic per disk) had larger
inhibition zones than disks with extracts mixed in other
ratios (Fig. 1).
A2C/proline assay. Previous studies in our laboratory have
provided indications that in animal cell and yeast model
systems, phenolics could modulate the cellular redox re-
TABLE 1. Total phenolic contents of oregano
and cranberry in various mixtures
Amt of phenolics
(mg/g, dry wt)
75% oregano, 25% cranberry ...................................................15.1
50% oregano, 50% cranberry ...................................................12.2
25% oregano, 75% cranberry ...................................................10.0
100% cranberry........................................................................... 7.4
VOL. 71, 2005H. PYLORI INHIBITION BY PHYTOCHEMICAL SYNERGIES8559
sponse through the proline-linked pentose phosphate path-
way (32). Therefore, the rationale for the A2C/proline assay
was to evaluate if small phenolics in the phytochemical
mixtures behave as proline analogs and, if so, could inhibit
proline dehydrogenase at the plasma membrane level in a
prokaryotic cell (which is associated with energy produc-
tion) and inhibit the bacterium. If this is the case, then only
addition of proline could overcome the inhibition of A2C or
A2C-linked phenolics with aromatic ring structures. In this
study, A2C, phenolics, and combinations of A2C and phe-
nolics enhanced the antimicrobial activity, and addition of
proline helped to overcome the inhibition by A2C and/or
phenolics, therefore indicating that site of action of pheno-
lics could be proline dehydrogenase.
Specifically, the antimicrobial effect of phytochemical ex-
tracts was further enhanced by adding 100 ?l of 0.1 mM A2C
to paper disks, and a larger inhibition zone was observed
(Fig. 2). Disks containing phytochemical extracts (at 0.1 mg
phenolics per disk) showed no inhibition when the plates con-
tained 1.0 mM proline, indicating that the antimicrobial effect
from phytochemicals was overcome by proline. When both
A2C and the phytochemical extract mixture were applied on
the plates containing 1.0 mM proline, a smaller inhibition zone
was observed than without proline, indicating that the antimi-
crobial effect was reduced in the presence of proline. Since
phytochemicals enhance the effect of A2C and respond to
proline similarly to A2C, these results provided clues that the
likely site of action of phenolic phytochemicals was proline
dehydrogenase. Future studies will further evaluate this en-
zyme response in detail.
Urease activity assay. Figures 3 and 4 show that the urease
activity was inhibited in the plates containing phytochemical
mixtures. The transition range of bromocresol purple is pH 5.2
to 6.8. The pH of the plates was 6.0 and the color was yellowish
prior to inoculation with H. pylori. After inoculation and 48 h
of incubation at 37°C, the area with bacterial growth turned
into purple, indicating that the bacteria were able to counter
the pH of the medium, likely by producing ammonia through
urease activity. When the paper disks containing extract mix-
tures were placed on the medium, the area around the paper
disks did not change to purple, indicating that the bacteria did
not counter the pH of medium and urease was likely inhibited.
The diameter of the yellow zone was measured and is shown in
Fig. 4. The mixture of oregano (25%) and cranberry (75%)
gave a larger yellow zone, indicating a higher inhibition of
FIG. 1. Antimicrobial activities of oregano and cranberry extract
mixtures against H. pylori at pH 7.0 and 37°C after 48 h of incubation
(100 ?l extract mixture per disk with 0.1 mg equivalent phenolic con-
tent). Each experiment consisted of three replicates with various phy-
tochemical concentrations. Each experiment was repeated three times.
The error bars represent ?1 standard deviation from the mean.
Bars with the same letters are not significantly different. Statistical
analysis was done by analysis of variance; P ? 0.05. Controls con-
sisted of disks with distilled water only.
FIG. 2. Synergistic effects of oregano and cranberry extracts on
inhibition of H. pylori in the presence of A2C/proline. Proline
(Sigma, St. Louis, MO) was added into the medium to a final
concentration of 1.0 mM. A2C was prepared at a concentration of
0.1 mM and added at 100 ?l to paper disks. The total phenolic
concentration was 0.1 mg/disk, and the inhibition diameter was
monitored after incubation at 37°C for 48 h. Controls were at the
same conditions with the same volume of water. O/C, oregano and
cranberry mixture at 25%/75%, wt/wt. The pH was 7.0. Each exper-
iment consisted of three replicates of various phytochemical con-
centrations. Each experiment was repeated three times. The error
bars represent ?1 standard deviation from the mean. Bars with the
same letters are not significantly different. Statistical analysis was
done by analysis of variance; P ? 0.05.
FIG. 3. Examples of urease inhibition in agar diffusion assay.
(A) Control. One hundred microliters of sterile water was added to the
paper disk. (B) One hundred microliters of phytochemical extracts was
added into the paper disk. All extract mixtures were tested at the level
of 0.05 mg phenolics per disk for urease inhibition. The plate medium
was adjusted to pH 6.0. The yellow zone indicates the area where there
is no urease activity. The purple zone indicates the area where urease
8560 LIN ET AL.APPL. ENVIRON. MICROBIOL.
The urease activity assay was also performed in liquid cul-
ture to determine whether the phytochemical extracts inhibited
the urease activity. H. pylori was treated with potential phyto-
chemical urease inhibitors prior to determining the urease
activity. Cells treated with 0.05 mg oregano phenolics/ml
showed a marked decrease in the urease activity (52% inhibi-
tion compared to the control). The mixture of phenolics in
extract also resulted in 29% inhibition. Cranberry phenolics
individually resulted in 9% urease inhibition compared to the
control (Fig. 5.) These results suggested that the phytochemi-
cals inhibited the urease activity. However, unlike in plate
assays, oregano alone had the most potent inhibitory activity,
which may be a reflection of differences in the types of phe-
nolics in cranberry and oregano and their behavior in a liquid
system compared to a solid system in a plate assay. It is likely
that oregano being enriched in partially hydrophobic phenolics
could effectively affect enzymes such as urease that are located
on the plasma membrane. As opposed to oregano, cranberry
has good acidifying and soluble phenolics that enter the cytosol
and are likely less effective on urease but more effective at the
proline dehydrogenase level.
Previous studies have indicated the antimicrobial potential
of phytochemical extracts (6, 7, 11). Oregano and cranberry are
useful botanicals which are generally recognized as safe for
food flavoring and as potential functional ingredients, which
are known for their antimicrobial activity linked to the phe-
nolic moiety. Phenolic phytochemicals such as ellagic acid and
rosmarinic acid have the potential to interact with proteins and
alter their conformation. These phytochemicals can directly
interact with the receptors on the cell membrane and could
affect normal functioning of ion pumps (17, 27, 28, 32, 39, 41).
Also, the partially hydrophobic nature of phenolic constituents
allows for accumulation and attachment in the bacterial cyto-
plasmic membrane, where inhibitory effects may eventually
lead to cell death. Recent evidence has also indicated that
altering multidrug resistance pumps on the cytoplasmic mem-
brane of bacteria by inhibitors or genetic knockout can en-
hance the antimicrobial function of phytochemicals (36).
Therefore, in oregano and cranberry phenolic profiles, specific
phenolics may inhibit multidrug resistance pumps, allowing
other phenolics in a synergistic profile to inhibit the bacterium.
Plate assay results indicated that the oregano and cranberry
extract mixture was superior in inhibiting H. pylori than indi-
vidual extracts at the same phenolic concentration. When dif-
ferent extract ratios based on phenolic content were used, a
larger inhibition zone was observed, indicating higher suscep-
tibility to a specific ratio (25% oregano and 75% cranberry) of
extract mixture. This may be due to one (or more than one)
specific phenolic present in the extract that damages the mem-
brane first, making cells more sensitive to the other phenolics
(36). As a consequence, impairment of proton pumps and loss
of H?-ATPase in damaged membranes can cause disruption in
the normal cellular function of the microorganism and there-
fore lead to cell death (Fig. 6). Further, the acidic nature of
phenolic-containing extracts themselves at higher concentra-
tions may create a low-pH microenvironment due to proton
donation and cell membrane disruption due to stacking (32),
which is likely more effective than low pH alone. Clues from
this study indicated that the mechanism of action for regulating
membrane-linked energy production could be through proline
dehydrogenase, based on the studies with A2C and phenolics
as well as combinations. These studies indicated that phenolics
in phytochemical extracts behaved similarly to the proline
analog A2C and that the inhibitory effect could be overcome by
proline. This provided clues that proline dehydrogenase at the
plasma membrane could likely be the site of action for phe-
Our results indicated that the activity of urease was in-
hibited in the presence of phytochemical extracts. The
mechanism of urease inhibition is not clear. It could be due
to the conformational change of urease by phenolics. In
FIG. 4. Synergistic effect of oregano and cranberry extracts on
inhibition of urease. The plate medium was adjusted to pH 6.0. The
total phenolic concentration was 0.05 mg/disk, and inhibition diam-
eter was monitored after incubation at 37°C for 48 h. The control
was a bacterial culture under the same conditions in which same
volume of water instead of phytochemical was placed. Each exper-
iment consisted of three replicates of various phytochemical con-
centrations. Each experiment was repeated three times. The error
bars represent ?1 standard deviation from the mean. Bars with
same letters are not significantly different. Statistical analysis was
done by analysis of variance; P ? 0.05.
FIG. 5. Synergistic effect of oregano and cranberry extracts on
inhibition of urease in broth. The total phenolic concentration was
0.05 mg/ml. The control was a bacterial culture under the same
conditions in which same volume of water instead of phytochemical
was placed. The data are means and standard deviations from
VOL. 71, 2005 H. PYLORI INHIBITION BY PHYTOCHEMICAL SYNERGIES 8561
FIG. 6. Proposed mechanism of antimicrobial effects of phenolic phytochemicals in prokaryotic cells. PPP, pentose phosphate pathway; SOD,
superoxide dismutase; CAT, catalase; GR, glutathione reductase; P-5-C, pyrroline-5-carboxylate; TCA, tricarboxylic acid; PRPP, phosphoribo-
sylpyrophosphate; PMF, proton motive force; H.p., H. pylori; O/C, oregano or cranberry.
plate assay, combinations of cranberry and oregano were
effective, indicating that higher-molecular-weight phenolics
from oregano may operate at the plasma membrane level
and subsequently, upon urease inhibition, small phenolics
from cranberry may affect cytosolic functions. On the other
hand, in the liquid system oregano phenolics were most
effective, as it is possible that partial hydrophobicity of phe-
nolics of oregano may allow attachment and inhibition at the
plasma membrane level.
In this study, the effects of a combined treatment with an
oregano and cranberry extract mixture on the inhibition of H.
pylori were demonstrated in plate assays and specific urease inhi-
bition was demonstrated in plate and broth assays. These combi-
nations of beneficial plant extracts provide a natural and dietary
solution, as well as an additional strategy to inhibit the growth of
H. pylori. Synergistic effects of combinations of plant extracts
provide a wide range of phenolic diversity, significantly increasing
antimicrobial efficacy. If the antimicrobial mechanisms at the cel-
lular level are further confirmed based on clues from this study,
then this is an excellent strategy to design the right plant extract
with a specific phenolic profile to prevent H. pylori infection. Also,
the diversity of phenolic types from different botanical sources
greatly increases the functionality for health and wellness (e.g.,
diseases). Such phenolic profiles also have the added benefit of
enhancing host tissue and cellular responses through enhanced
antioxidant enzyme activity (32).
The exact mechanisms of cellular damage by phenolics at
the urease or proline dehydrogenase level will be further
investigated in our laboratory. If these mechanisms are fur-
ther elucidated, designing specific phenolics profiles to in-
hibit H. pylori will become feasible and also provide addi-
tional health benefits.
1. Akiyama, H., K. Fujii, O. Yamasaki, T. Oono, and K. Iwatsuki. 2001. Anti-
bacterial action of several tannins against Staphylococcus aureus. J. Antimi-
crob. Chemother. 48:487–491.
2. Akyon, Y., and G. Hascelik. 1999. The effect of Helicobacter pylori on neu-
trophil chemotaxis is independent of caga. FEMS Immunol. Med. Microbiol.
3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye bind-
ing. Anal. Biochem. 72:248–254.
4. Cammarota, G., V. Pasceri, A. Papa, R. Cianci, A. Gasbarrini, P. Fedeli, F.
Cremonini, G. Fedeli, A. Maseri, and G. Gasbarrini. Helicobacter pylori infec-
tion and ischaemic heart disease. J. Gastroenterol. Hepatol. 30:304–306.
5. Chandler, S. F., and J. H. Dodds. 1983. The effect of phosphate nitrogen and
sucrose on the production of phenolics and socosidine in callus cultures of
Solanum tuberosum. Plant Cell Rep. 2:105–108.
6. Chun, S. S., D. A. Vattem, Y.-T. Lin, and K. Shetty. 2005. Phenolic antioxi-
dants from clonal oregano (Origanum vulgare) with antimicrobial activity
against Helicobacter pylori. Process Biochem. 40:809–816.
7. Cowan, M. M. 1999. Plant products as antimicrobial agents. Clin. Microbiol.
8. Creno, R. J., R. E. Wenk, and P. Bohlig. 1970. Automated micromeasure-
ment of urea using urease and the Berthelot reaction. Am. J. Clin. Pathol.
9. Danesh, J., and R. Peto. 1998. Risk factors for coronary heart disease and
infection with Helicobacter pylori: meta-analysis of 18 studies. Br. Med. J.
10. Daroch, F., M. Hoeneisen, and C. L. Gonzalez. 2001. In vitro antibacterial
activity of Chilean red wines against Helicobacter pylori. Microbios 104:79–85.
11. Dean, S. G., and G. Richie. 1987. Antimicrobial properties of plant essential
oils. Int. J. Food Microbiol. 5:165–180.
12. De Boer, W. A., and G. N. Tytgat. 2000. Treatment of Helicobacter pylori
infection. Br. Med. J. 320:31–34.
13. Dunn, B. E., H. Cohen, and M. J. Blaser. 1997. Helicobacter pylori. Clin.
Microbiol. Rev. 10:720–741.
14. Gene, E., X. Calvet, R. Azagra, and J. P. Gisbert. 2003. Triple vs. quadruple
therapy for treating Helicobacter pylori infection: a meta-analysis. Aliment.
Pharmacol. Ther. 17:1137–1143.
15. Hu, L. T., and H. L. T. Mobley. 1990. Purification and N-terminal analysis of
urease from Helicobacter pylori. Infect. Immun. 58:992–998.
16. Jones, N. L., S. Shabib, and S. M. Sherman. 1997. Capsaicin as an inhibitor
of the growth of the gastric pathogen Helicobacter pylori. FEMS Microbiol.
17. Kim, H. J., K. S. Yum, J. H. Sung, D. J. Rhie, M. J. Kim, D. S. Min, S. J.
Hahn, M. S. Kim, Y. H. Jo, and S. H. Yoon. 2004. Epigallocatechin-3-gallate
increases intracellular [Ca2?] in U87 cells mainly by influx of extracellular
Ca2? and partly by release of intracellular stores. Naunyn Schmiedebergs
Arch. Pharmacol. 369:260–267.
18. Kouassi, Y., and L. A. Shelef. 1998. Inhibition of Listeria monocytogenes by
cinnamic acid—possible interaction of the acid with cysteinyl residues. J.
Food Saf. 18:231–242.
19. Larson, A. E., R. R. Y. Yu, O. A. Lee, S. Price, G. J. Haas, and E. A. Johnson.
1996. Antimicrobial activity of hop extracts against Listeria monocytogenes in
media and in food. Int. J. Food Microbiol. 33:195–207.
20. Lin, Y.-T., R. G. Labbe, and K. Shetty. 2004. Inhibition of Listeria monocy-
togenes in fish and meat systems using oregano and cranberry synergies.
Appl. Environ. Microbiol. 70:5672–5678.
21. Mendall, M. A., P. M. Goggin, N. Molineaux, J. Levy, T. Toosy, D. Stachan,
A. J. Camm, and T. C. Northfield. 1994. Relation of Helicobacter pylori
infection and coronary heart disease. Br. Heart J. 71:437–439.
22. Moayyedi, P., S. Soo, J. Deeks, D. Forman, J. Mason, and M. Innes. 2000.
Systematic review and economic evaluation of Helicobacter pylori eradication
treatment for non-ulcer dyspepsia. Br. Med. J. 321:659–664.
23. Murray, L. J. 1997. Helicobacter pylori infection and ischaemic heart disease.
24. Murray, L. J., A. J. Lane, I. M. Harvey, J. L. Donovan, P. Nair, and R. F.
Harvey. 2002. Inverse relationship between alcohol consumption and active
Helicobacter pylori infection: the Bristol Helicobacter project. Am. J. Gastro-
25. Nagata, K., T. Mizuta, Y. Tonokatu, Y. Fukuda, H. Okamura, T. Hayashi, T.
Shimoyama, and T. Tamura. 1992. Monoclonal antibodies against the native
urease of Helicobacter pylori: synergistic inhibition of urease activity by
monoclonal antibody combinations. Infect. Immun. 60:4826–4831.
26. Nakamura, H., H. Yoshiyama, H. Takeuchi, T. Mizote, K. Okita, and T.
Nakazawa. 1998. Urease plays an important role in the chemotactic
motility of Helicobacter pylori in a viscous environment. Infect. Immun.
27. Pan, C. Y., Y. H. Kao, and A. P. Fox. 2002. Enhancement of inward Ca (2?)
currents in bovine chromaffin cells by green tea polyphenol extracts. Neuro-
chem. Int. 40:131–137.
28. Papadopoulou, A., and R. A. Frazier. 2003. Characterization of protein—
polyphenol interactions. Trends Food Sci. Technol. 5:186–190.
29. Parente, F., G. Maconi, O. Sangaletti, M. Minguzzi, L. Vago, E. Rossi, and
P. G. Bianchi. 1996. Prevalence of Helicobacter pylori infection and related
gastroduodenal lesions in spouses of Helicobacter pylori positive patients with
duodenal ulcer. Gut 39:629–633.
30. Seaberg, A., R. L. Labbe, and K. Shetty. 2003. Inhibition of Listeria mono-
cytogenes by elite clonal extracts of oregano (Origanum vulgare). Food Bio-
31. Shetty, K., O. F. Curtis, R. E. Levin, R. Witkowsky, and W. Ang. 1995.
Prevention of vitrification associated with in vitro shoot culture of oregano
(Origanum vulgare) by Pseudomonas spp. J. Plant Physiol. 147:447–451.
32. Shetty, K., and M. L. Wahlqvist. 2004. A model for the role of proline-linked
pentose phosphate pathway in phenolic phytochemical biosynthesis and
mechanism of action for human health and environmental applications. Asia
Pacific J. Clin. Nutr. 13:1–24.
33. Smoot, D. T., H. L. T. Mobley, G. R. Chippendale, J. F. Lewison, and J. H.
Resau. 1990. Helicobacter pylroi urease activity is toxic to human gastric
epithelial cells. Infect. Immun. 58:1992–1994.
34. Somal, A. N., K. E. Coley, P. C. Molan, and B. M. Hancock. 1994. Suscep-
tibility of Helicobacter pylori to the antibacterial activity of manuka honey.
J. R. Soc. Med. 87:9–12.
35. Stevenson, T. H., L. M. Lucia, and G. R. Acuff. 2000. Development of a
selective medium for isolation of Helicobacter pylori from cattle and beef
samples. Appl. Environ. Microbiol. 66:723–727.
36. Tegos, G., F. R. Stermitz, O. Lomovskaya, and K. Lewis. 2002. Multidrug
pump inhibitors uncover remarkable activity of plant antimicrobials. Anti-
microb. Agents Chemother. 46:3133–3141.
VOL. 71, 2005H. PYLORI INHIBITION BY PHYTOCHEMICAL SYNERGIES 8563
37. Tougas, G., Y. Chen, P. Hwang, M. M. Liu, and A. Eggleston. 1999.
Prevalence and impact of upper gastrointestinal symptoms in the Cana-
dian population: findings from the digest study. Am. J. Gastroenterol.
38. Tsuchiya, H. 2001. Biphasic membrane effects of capsaicin, an active com-
ponent in Capsicum species. J. Ethnopharmacol. 75:295–299.
39. Tsuchiya, H., M. Sato, Y. Kameyama, N. Takagi, and I. Namikawa. 1987.
Effect of lidocaine on phospholipid and fatty acid composition of bacterial
membranes Lett. Appl. Microbiol. 4:141–144.
40. Tsuda, M., M. Karita, M. G. Morshed, K. Okita, and T. Nakazawa. 1994. A
urease-negative mutant of Helicobacter pylori constructed by allelic exchange
mutagenesis lacks the ability to colonize the nude mouse stomach. Infect.
41. Vattem, D. A., Y.-T. Lin, R. Ghaedian, and K. Shetty. 2005. Cranberry
synergies for dietary management of Helicobacter pylori infections. Process
42. Xu, J. K., C. S. Goodwin, M. Cooper, and J. Robinson. 1990. Intracellular
vacuolization caused by the urease of Helicobacter pylori. J. Infect. Dis.
43. Zheng, Z., and K. Shetty. 2000. Solid-state bioconversion of phenolics from
cranberry pomace and role of Lentinus edodes ?-glucosedase. J. Agric. Food
8564 LIN ET AL.APPL. ENVIRON. MICROBIOL.