[email protected] (4)

8/17/2019 [email protected] (4)

1/5

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: [email protected]

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


2/5

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-


3/5

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-


4/5

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.

References

l11

Beretz, A. and Cazenave. J.P. (1991) Old and new natural

products as the source of modern antithrombotic drugs. Planta

Med. 57, S68-S72.

t21

Bolton, S.. Null, G. and Troetel. W.M. (19821 The medical

uses of garlic-fact and fiction, J. Am. Pharmacol. Ass. 22.

40-43.

131

On the other hand, Deshpande et al. demonstrated

that AGE in an acidic buffer (pH 2.0) did not show

any loss of antibacterial activity [7].

Insensee, H.. Rietz, B. and Jacob, R. (19931 Cardioprotective

actions of garlic

Allium satic,um).

Arzneimittelforschung 43.

94-98.

t41

The antibacterial activity of AGE against H. py-

lo r i was assessed by our data and concentrations

lower than 5 mg ml-’ were able to inhibit the

strains. In most cases the inhibitory concentrations

were also bactericidal. In fact, the MBC values were

equal or two-fold higher than MICs.

Lawson, L.D.. Ransom, D.K. and Hughes, B.G. t 19921 Inhi-

bition of whole blood platelet aggregation by compounds in

garlic clove extracts and commercial garlic products. Thromb.

Res. 65. 141-156.

El

Cavallito. C.J.. Buck, S.J. and Suter, C.M. (19441 Allicin, the

antibacterial principle of A urn saticum. I. Isolation. physi-

cal properties and antibacterial action. J. Am. Chem. Sot. 66,

1950-1951.

161

One interesting finding is that heated garlic ex-

tract was also effective. Indeed. the antibacterial

properties are reduced only in one or two values.

MIC and MBC values obtained with boiled AGE

were two- to four-fold higher than MIC and MBC

values obtained with fresh AGE.

[71

181

Furthermore, 10 g of fresh garlic yielded 1.6 &

0.05 g of AGE and one clove (mean weight 1.59 g)

contained 0.26 g of AGE; therefore even small

amounts of garlic (one clove) will be highly effective

in promoting antibacterial activity against

H. pylori.

Moreover, in our data we found a synergistic

effect of garlic combined with omeprazole. In nine

strains (47%) the combination of drugs showed a

FIC I 0.5. The combination of drugs did not show

antagonistic effect.

[91

[lOI

[I II

[I21

l131

l141

[I51

Delaha. EC. and Garagusi. V.F. (19851 Inhibition of My-

cobacteria by garlic extract Allium

scrtitwm).

Antimicrob.

Agents Chemother. 27, 485-486.

Deshpande. R.G., Khan, M.B.. Bhat, D.A. and Navalkar.

R.G. (19931 Inhibition of

M~cohacterium arium

complex

isolates from AIDS patients by garlic AIIium

sutir~um).

J.

Antimicrob. Chemother. 32, 623-626.

Gonzalez-Fandos. E., Garcia-Lopez. M.L.. Sierra, M.L. and

Otero. A. (19941 Staphylococcal growth and enterotoxins

(A-D) and thermonuclease synthesis in the presence of

dehydrated garlic. J. Appl. Bacterial. 77. 549-552.

Wills. E.D. (19561 Enzyme inhibition by allicin, the active

principle of garlic. Biochem. I. 63, 5 14-520.

Amer. M., Taha. M. and Tosson, Z. t 19801 Effect of aqueous

garlic extract on the growth of dermatophytes. Intern, J.

Dermatol. 197. 285-287.

Ghannoum. M.A. (19881 Studies on the anticandidal mode of

action of

Allium scitiwm

(garlic). J. Gen. Microbial. 134.

29 17-2924.

This report suggests that in vitro laboratory find-

ings on the bacteriostatic and bactericidal activity of

AGE against

H. pylori

could have applications in in

vivo conditions.

Pai. S.T. and Plan, M.W. t 19951 Antifungal effect of

Allium

.scrtirum

(garlic) extract against the

Aspergillus species

in-

volved in otomycosis. Lett. Appl. Microbial. 20, 14-18.

Amonkar. S.V. and Banereji, A. (1971) Isolation and charac-

terization of larvicidal principle of garlic, Science 174.

1343- 1344.

Acknowledgements

This work was supported by grant no.

CU.93.04315 from the Consiglio Nazionale delle

Soffar. S.A. and Mokhtar, G.M. (19911 Evaluation of the

antiparasitic effect of aqueous garlic ( Alhm satkum) extract

in hymenolepiasis nana and giardiasis. J. Egypt. Sot. Para-

sitol. 2 I. 497-502.

Cavallito. C.J. and Bailey, J.H.(19441 Allicin. the antibacte-

rial principle of Allium satirum. II. Dermination of the

chemical structure. J. Am. Chem. Sot. 66, 1952-1954.


5/5

L. Cellini et al. /FEMS Immunolq~y cmd Medical Microhiolog~ 13 1996) 273-777

371

1161 Feldberg. R.S.. Chang, S.C.. Kotik, A.N., Nadler, M..

Neuwirth. Z.. Sundstrom, D.C. and Thompson, N.H. (1988)

In vitro mechanism of inhibition of bacterial cell growth by

allicin. Antimicrob. Agents Chemother. 32, 1763-1768.

[171 Lee, A. 1994) The microbiology and epidemiology of He/i-

cohoctrr phri infection. Stand. J. Gastroenterol. 29 (Suppl.

201), 2-6.

[I81 Marshall. B.J. (1983) Unidentified curved bacilli on gastric

epithelium in active chronic gastritis. Lancet i. 1273-1275.

[

191 Marshall. B.J. (1994)

Helicobactrr pylori.

Am. J. Gastroen-

terol. 89. Sl l6-Sl22.

[20] Parsonnet. J.. Friedman, G.D., Vandersteen. D.P., Chang. Y..

Vogelman. J.H.. Orentreich, N. and Sibley, R.K. (1991)

Hrlicobactrr p,vlori infection and the risk of gastric carci-

noma. N. Engl. J. Med. 325. 1127-1131.

[21] Burette, A. and Glupczynski. Y. (1995) Helicobacter pylori:

the place of the new macrolides in the eradication of the

bacteria in peptic ulcer disease. Infection 23. S44-S52.

[22] Bell, G.D.. Powell. K.U.. Burridge, S.M., Bowden. A.F..

Atoyebi. W., Bolton, G.H.. Jones. P.H. and Brown, C. (1995)

Rapid eradication of Helicobacter phri infection. Aliment.

Pharmacol. Ther. 9. 41-46.

[23] Cellini. L.. Marzio. L.. Di Girolamo, A., Allocati. N.. Grossi,

L. and Dainelli. B. (1991) Enhanced clearing of

Helicobac-

ter pvlori after omeprazole plus roxitromycin treatment.

FEMS Microbial. Lett. 83. 255-258.

[24] Graham. D.Y., Lew, GM., Malaty. H.M.. Evans, D.G..

Evans, D.J. Jr.. Klein. P.D.. Alpert. L.C. and Genta, R.M.

(1992) Factors influencing the eradication of Helicobactrr

pylori

with triple therapy. Gastroenterology 102, 493-496.

(251 Cellini, L., Allocati, N.. Piccolomini, R., Di Campli. E. and

Dainelli. B. 1992) New plate medium for growth and detec-

tion of urease activity of Helicobacrrr pxlori. J. Clin. Micro-

biol. 30. 1351-1353.

[26] Hazel], S.L. (1993) Cultural techniques for the growth and

isolation of Helicohacter pylori. In: Helicohacter pylori:

biology and clinical practice (Goodwin. C.S. and Worsleq.

B.W.. Eds.). pp. 373-283. Boca Raton Florida. CRC Press.

[27] Drumm. B. and Sherman. P. (1989) Long-term storage of

Campylohactrr pylori. J. Clin. Microbial. 27. 1655-1656.

[28] National Committee for Clinical Laboratory Standards.( 1990)

Methods for dilution antimicrobial susceptibility tests for

bacteria that grow aerobically. Approved standard M7-A2.

Nat. Comm. Clin. Lab. Standards Villanova. PA.

[29] Piccolomini. R.. Di Girolamo. A.. lacone. A., Fioritoni, C..

Cellini. L.. Nicoletti, M. and D‘Antonio. D. (1991) Clinical

efficacy and in vitro activity of the ciprofloxacin-azlocillin

combination (ratio I : IO) against Gram-negative bacteria from

non neutropenic hematologic patients. Drugs Exp. Clin. Res.

17 (9). 455-460.

Documents

[email protected] (4)