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IMMUNOLOGY AND
MEDICAL
MICROBIOLOGY
LS VI R
FEMS Immunology and Medical Microbiology 13 1996) 273-277
Inhibition of Helicobacter pylori by garlic extract
Allium sativum)
Luigina Cellini *
Emanuela Di Campli, Michele Masulli, Soraya Di Bartolomeo,
Nerino Allocati
Received I6 August 1995: revised 20 November 1995: accepted 2
I
November I995
Abstract
The antibacterial effect of aqueous garlic extract (AGE) was investigated against
Helicobacter pylori.
Sixteen clinical
isolates and three reference strains of H. pylori were studied. Two different varieties of garlic were used. The concentration
of AGE required to inhibit the bacterial growth was between 2-5 mg ml- ‘.
The concentration, for both AGE types, to
inhibit 90% (MIC,,) of isolates was 5 mg ml-‘. The minimum bactericidal concentration (MBC) was usually equal to, or
two-fold higher than, minimum inhibitory concentration (MIC). Heat treatment of extracts reduced the inhibitory or
bactericidal activity against
H. pyl on’; the
boiled garlic extract showed a loss of efficacy from two- to four-fold the values of
MIC and the MBC obtained with fresh AGE. The antibacterial activity of garlic was also studied after combination with a
proton pump-inhibitor (omeprazole) in a ratio of 25O:l. A synergistic effect was found in
47
of strains studied: an
antagonistic effect was not observed.
k’e~~~wrrl\c Hrlicr~hrrctrr
pdori; Aqueous
garlic extract: Minimum inhibitory concentration: Minimum bactericidal concentration: Omepra-
zole
1 Introduction
[9] specified that allicin is an inhibitor of sulphydryl
The long history of the medicinal use of garlic is
well-documented: the properties of garlic against
atherosclerosis, coronary thrombosis
[l-3],
inhibi-
tion of platelet aggregation [4] and much information
on its antibacterial [5-91, antifungal [lo-121 and
antiprotozoal [ 13,141 properties are widely known.
In 1944. Cavallito and Bailey identified diallyl
thiosulfinate (allicin) as one of the agents responsible
for the antimicrobial activity of garlic [15,16]. Wills
metabolic enzymes and reported that the antimicro-
bial properties are due to specific interference with
SH-groups.
Helicobacter pylon is a fastidious Gram-negative,
curved rod which is associated with active chronic
gastritis and gastroduodenal ulcer disease and in the
development of gastric cancer [ 17-201. Although
H
pylor i is sensitive to a large number of antimicrobial
agents in vitro [21]. the effectiveness in vivo has
been disappointing as most antimicrobials have
”
Corresponding author. Tel: + 39 (871) 355279: Fax: + 39
(871) 355282: E-mail: CELLINI@JIMS.UNICH.IT
proven ineffective in the eradication of this strain.
Several therapeutic regimens have been proposed to
clear
H ylor i
infection and, in particular. combina-
0928.824~/96/ 15.00 Q 1996 Federation of European Microbiological Societies. All rights reserved
.SSD/ 0928.8241(95 100 120-J
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214 L. Cell ini et al. FEM S Immunology and M edical M icrobiol ogy 13 C 996173-277
tions of drugs have been shown to be effective
passed through a 0.45 pm filter (Millipore, SpA,
[22-241. In these studies the eradication of the or-
Rome, Italy), sterilized through a 0.22 pm Millipore
ganism from the gastric environment was assessed in filter (Millipore) and kept frozen at - 70°C and
long-term controls.
lyophilized.
In the present study the effect of garlic extract
either alone when used fresh or boiled, or combined
with a proton pump-inhibitor, omeprazole, on the
growth and survival of H. pylori was investigated.
From the two varieties of fresh garlic used (10 g)
the yield dry weight was 1.6 + 0.05 g AGE. The
fresh garlic was used immediately following the
extraction.
2.3. In Litro susceptibility tests
2. Materials and methods
2 I Bacteria and preparation of inocula
Sixteen strains of H. pylori isolated from antral
mucosal biopsies of patients with chronic gastritis or
duodenal ulcer were used; the strains were identified
on the basis of the colony appearance, Gram-stain-
ing, and positive reactions in biochemical tests
(catalase, urease, and oxidase) [25,26]. All strains
were stored for long-term maintenance, at - 70°C
[27].
H. pylori
ATCC 43579,
H. pylori
ATCC
43504, H. pylori ATCC 43629 were used as stan-
dard control strains. StaphyZococcus aureus ATCC
29213 and
Escherichia coli
ATCC 25922 were used
as controls.
The minimum inhibitory concentration (MIC) was
determined, in duplicate, by an agar dilution method
following the National Committee for Clinical Labo-
ratory Standards guidelines [28] using Mueller-Hin-
ton agar (Biolife Italiana) with 7% (v/v) laked horse
blood and a multipoint inoculator, delivering 1 ~1 of
bacteria suspension (5 X 105/spot) prepared as de-
scribed above. Omeprazole (Hassle Astra, Sweden),
reconstituted following the manufacturer’s instruc-
tions, was used in serial two-fold dilutions ranging
from 2-64 pg ml-‘.
AGE was tested at serial
twofold dilutions in two ranges from 0.25-8.0 mg
ml-’ and 0.3-10 mg ml-‘.
The activity of omeprazole combined with AGE
Strains were cultured in duplicate on non-selec-
tive medium containing Columbia agar base (Un-
ipath) with 10% (v/v> laked horse blood plus IsoVi-
taleX 1% (v/v> (CA) (BBL, Microbiology System)
and incubated in a microaerophilic environment at
37°C for five days (GasPak, BBL). The bacterial
suspensions were prepared in Brucella broth and
adjusted to a McFarland standard No. 5 (approxi-
mately 5 X lo9 cfu ml- ‘).
was evaluated in the ratio 1:250. Plates were pre-
pared immediately prior to inoculation. The MIC
was read three days after incubation at 37°C in the
microaerophilic atmosphere. MIC was defined as the
lowest concentration of drug inhibiting visible bacte-
rial growth.
2.2. Preparation of aqueous garlic extract (AGE)
Bulbs of garlic (A. satiuum L.) were purchased
from a local store. Two different varieties of garlic,
with red pellicle or white pellicle, were used. Fresh
cloves of garlic (100 g> were peeled, minced and
ground in a blender. The garlic pulp was agitated
with 250 ml of phosphate buffer, 10 mM pH 7.0, on
a shaker for 1 h and refrigerated for 2 h. The product
was squeezed through gauze cloth to remove the
larger particles and centrifuged at 2500 X g for 45
min. The supemate of garlic extract (AGE) was
To evaluate the effect of the omeprazole com-
bined with AGE, the fractional inhibitory concentra-
tion index (FIG) [29] was calculated. FIC indices
were calculated according to the following formula:
FIC index = (MIC of drug A in combination/MIC
of drug A alone) + (MIC of drug B in
combination/MIC of drug B alone 1. An FIC index
I 0.5 indicated a synergistic combination, an index
2 2 indicated antagonism, whereas values of 0.5-2
represented an additive effect. To determine the min-
imum bactericidal concentration (MBC) the broth
MIC was carried out with Brucella broth plus 2% of
fetal calf serum. This test was done by broth mi-
crodilution procedures with a final inoculum of about
5 X 1OS cfu ml ’ After incubation for three days at
37°C in microaerophilic atmosphere, the MIC was
defined as the lowest drug concentration that inhib-
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ited visible growth in broth. The MBC was deter-
mined, in duplicate, by subculturing 100 ~1 of broth
without visible growth onto CA without drugs. The
MBC was defined as the lowest concentration of
drug which killed the 99.9% of the original inoculum
after three days of incubation at 37°C in a controlled
atmosphere.
To assess the stability of AGE when boiled, all
tests were prepared in duplicate, using one sample of
fresh AGE and one heated for 10 min at 100°C.
3. Results
The MIC and MBC values obtained for both
varieties of garlic against the 19 strains of H. pylori
are shown in Table 1.
The concentration of AGE required to inhibit H.
phi strains, was between 2.5-5.0 mg ml-‘. The
MIC at which 90% (MIC,,,) of the isolates were
inhibited was 5.0 mg ml-’ for both AGE types. The
concentration of AGE required to obtain a bacterici-
dal effect against H. pylori were the same or two-fold
higher than the corresponding MIC value. There
Table I
In vitro antihactrrial activity (mg ml-’ ) of AGE (red variety (a)
and white variety (h)) against 19 Hrlicohactrr /qki strains
H. pxlori MIC MBC MIC MBC
strainr (mgml-‘) (mgm- ‘) (mgml-‘) (mgml-‘I
(>I) (a)
(h) (b)
13 A 2.5 2.5 2 ?.S
18 5 5 15 4
21
B 1 5 5 5
28 25 2.5 5 5
52
BM 5 5 4 s
5M s 5 5 5
3M 5 5 1 5
IG 4 4 5 5
21 A 5 5 2.5 5
IX B 1 4 5 5
‘M 2 2 7 4
1M 4 4 2 1
6M 4 -1 4 1
31 A 1 2 2.5 2.5
IA 3 5 1 5
68 2 1.5 2.5 2.5
ATCC13501 4 1 3 5
ATCC43629 4 5 5 5
ATCC13.579 4 5 1 5
Table 7
In vitro combined activity of Omeprazole plus AGE ” (ratio
I :250) against 19
h strains of Hrlicohncrrr p~?ori
Combination No. of isolates (c/r) showing FIG index
(ratio I :? I)
syner- indiffer- antago- median range
pism ence
nism
OME + AGE 9(37)
lo(53) 0
0.67
0.16-1.3
” Mixture of AGE hy white and red garlic.
h Sixteen clinical isolates and 3 standard reference strains
were no significant differences between the MlCs
and MBCs with AGE obtained from either the white
or the red garlic.
The in vitro activity of omeprazole alone. against
the 19 H. p~lori studied, ranged from 2-32 pg
ml-‘; the MIC5,, (the MIC at which 507~ of the
isolates were inhibited) and the MIC,(, values were
16 pg mll’
and 32 pg mll’ respectively.
The effect of the combination of omeprazole plus
AGE in the ratio 1:250 is shown in Table 2.
The combination of omeprazole plus AGE exhib-
ited a synergistic effect against 9 to 19 (47%) strains
tested (the FIC index was lower than 0.51. For the
remaining strains, the association of drugs showed an
additive effect (FIG index values of 0.52). The
combination did not show an antagonistic effect (FIG
index 2 21.
The boiled garlic extract retained the antibacterial
activity, even though this effect was reduced. All the
MICs and the MBCs were two- to four-fold higher
than the respective MICs and MBCs obtained with
fresh AGE; with one strain H. pylori A22). the
boiled AGE exhibited an MIC eight-fold higher than
MIC obtained with fresh AGE. E. cwli ATCC 25922
and S. RUWUS ATCC 29213 were not inhibited by
the concentrations of AGE used in this work.
4. Discussion
There has been a renewed interest in the anti-in-
fective properties of medicinal plants and foods.
Garlic. in particular, is widely utilized as a folk
remedy for its antimicrobial and other beneficial
effects. The inhibitory activity of AGE against many
microorganisms has been mentioned in clinical re-
ports for nearly 100 years. and its use can be particu-
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276
L. Cellini et al. / FEMS Immunolop und Medical Microbiology 13
C
996) 273-277
larly important when traditional therapies are found
Ricerche, and from the Minister0 dell’Universit’a e
to be too toxic [12]. della Ricerca Scientifica e Tecnologica (60%).
All antimicrobial agents used against
H. pylori
were active in vitro at low concentrations, but the in
vivo eradication of the strain produced disappointing
results and the high number of
H. pylori
reinfection
in the gastric mucosa suggested incomplete eradica-
tion. These discrepancies between the in vitro and in
vivo results were probably due to marked differences
between the laboratory and in vivo environment. One
of the factors responsible for the difference is the
low pH of the stomach which reduces the antimicro-
bial activity of many drugs.
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