a Department of Medical Microbiology, University Hospital Maastricht; b Department of Epidemiology and University of Maastricht, The Netherlands c Department of Health Risk Analysis and Toxicology, University of Maastricht, The Netherlands
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Abstract |
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Introduction |
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For eradication of H. pylori, good results are obtained with a triple therapy consisting of the gastric acid inhibitor omeprazole and two antibiotics such as metronidazole, amoxycillin or clarithromycin. In vitro, an antibacterial effect of omeprazole on bacterial growth including H. pylori has been demonstrated. 2,3 As resistance of H. pylori to metronidazole, clarithromycin and tetracycline increases, reduction in the use of antibiotics is important to control the problem of antibiotic resistance.
We hypothesized that a possible synergic effect of garlic and antibiotics and garlic and omeprazole could result in a reduction in the use of antibiotics.
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Materials and methods |
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Fresh Spanish garlic was peeled and mashed in a blender. After filtration, the substance was freeze-dried and stored at -70°C until use. Commercial garlic tablets (Arizona 2000 TR, Kneipp and Wapiti 2000) kindly supplied by Nutrivital BV (Dronrijp, The Netherlands), Kneipp Nederland BV (Montfoort, The Netherlands) and Emonta BV (Drachten, The Netherlands) were pulverized with a mortar. Both the raw and commercial garlic tablets were dissolved in sterile distilled water before use.
Pure omeprazole, kindly supplied by Astra (Hässle, Sweden), was dissolved in 96% acidified ethanol, 10- 15 min before use to assure acid-catalysation.
Antibacterial effect of garlic on H. pylori
Susceptibility of H. pylori for garlic was determined by the agar dilution method using blood agar plates in miniwell format (Blood agar base no. 2 (Oxoid CM271, Basingstoke, UK) with 7% sheep blood) containing raw garlic extract or commercial garlic in concentrations of 0, 1000, 2000, 5000, 10,000, 12,500, 15,000 and 17,500 mg/L. Agar plates were inoculated with 0.01 mL of a H. pylori suspension (McFarland 3) and incubated for 4 days at 37°C under microaerophilic conditions (5% O 2, 10% CO 2). MIC values were also determined based on the allicin contents of the commercial garlic tablets.
The amount of thiosulphinates in the raw garlic extract was determined spectrophotometrically. Allicin is the major thiosulphinate produced by garlic and can be estimated by multiplying the value for total thiosulphinates by 0.7.
Combined influence of garlic and antibiotics
The combined effect of the home-made garlic extract and amoxycillin, clarithromycin and metronidazole on H. pylori was tested with blood agar plates containing either garlic 2000 mg/L alone or in combination with a quarter, once or twice the MIC values of amoxycillin (0.032 mg/L), clarithromycin (0.016 mg/L) or metronidazole (1 mg/L). Agar plates were inoculated with 0.1 mL of H. pylori suspension (McFarland 3) and incubated as described above.
Combined influence of garlic and omeprazole
The combined effect of garlic and omeprazole on H. pylori was tested with killing curves using Brain Heart Infusion broth pH 5 (Oxoid CM225) with 5% sheep blood. Combinations of garlic (500, 1000, 2000 or 4000 mg/L) and omeprazole (100 or 200 mg/L) as well as a suspension of H. pylori (inoculum size 10 5 cfu/mL) were added. From day 0 to day 7, a sample of 0.1 mL was taken and bacterial counts were assessed semi-quantitatively with the five segments method.
A control of bacterial growth and a control with acidified ethanol was included each time. All experiments were performed at least in duplicate.
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Results |
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Five clinical isolates of H. pylori were tested in triplicate. The median MIC values were 10,000 (range 5000- 10,000) mg/L for the raw garlic extract, 12,500 (range 10,000- 12,500) mg/L for Kneipp, 15,000 (range 12,500- 15,000) for Wapiti 2000 and >17,500 for Arizona 2000 TR. The allicin concentration is 0.77, 2.67 and 0.90 mg/tablet (including the coating) for Arizona 2000 TR, Kneipp and Wapiti 2000, respectively. For raw garlic extract the concentration of allicin determined spectrophotometrically was 12.3 mg/g garlic extract. If MIC values were based on the allicin contents of the commercial garlic tablets, MIC values for the three commercial garlic tablets were similar (40,000 mg/L).
As the antibacterial effect was strongest for the raw garlic extract and to circumvent a possible influence of the coating of the commercial garlic tablets, further experiments were performed with the raw garlic extract.
Combined effect of garlic and antibiotics
No synergic or antagonistic effect could be observed between garlic and amoxycillin, clarithromycin or metronidazole on H. pylori.
Combined effect of garlic and omeprazole
The combined effect of garlic and omeprazole on the growth of H. pylori tested with killing curves is shown in the Figure. Values are based on the mean of five clinical isolates tested in duplicate. Control growth of H. pylori varied between 10 4 and 10 7 cfu/mL during 7 days. In the presence of omeprazole (200 mg/L), garlic 2000 or 4000 mg/L, or combinations of both (see Figure, part a), no growth of H. pylori was observed after 24- 48 h. In the presence of omeprazole 100 mg/L (see Figure, part b), the number of H. pylori decreased slowly to zero within 7 days and a slight decrease of H. pylori was found in the presence of garlic 500 mg/L during the first 24- 48 h. In the presence of garlic 1000 mg/L the number of H. pylori decreased to 0- 10 1 cfu/mL during the first 3 days but rose again thereafter. A synergic effect was found when both omeprazole 100 mg/L and garlic 500 or 1000 mg/L were added, showing no bacterial growth after 24- 48 h.
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Discussion |
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Several in-vitro studies have shown an antibacterial effect of allium vegetables. 4 The antibacterial activity varies with the species, production method and ripeness. 5 All experiments in this study were performed with one batch of garlic extract and commercial garlic tablets. An antibacterial effect of garlic was reported previously for both Gram-positive and Gram-negative bacteria, including antibiotic-resistant strains. 4 Recently, two studies also described an antibacterial effect of garlic against H. pylori. 6,7
Garlic, like all allium vegetables, contains a wide range of thiosulphinates such as allicin which are thought to be responsible for the antibacterial activity. 4 Selective removal of the thiosulphinates or prevention of their formation by inhibition of alliinase eliminates the antibacterial activity of garlic. 4 When the commercial garlic tablets tested in this study were compared based on their allicin contents, no differences in antibacterial activity were found. Furthermore, the strongest antibacterial effects were observed for the raw garlic extract, which was found to have the highest concentration of allicin, supporting an important role for allicin in the antibacterial effects of garlic.
Theoretically, both thiosulphinates and omeprazole (a substituted benzimidazole) may have a similar mechanism for their antibacterial activity. As described for omeprazole, allicin can also bind to SH-groups of, for example, bacterial enzymes 8 and nutrients such as cysteine. 3,9 Competition between garlic and omeprazole for binding to available SH-groups may contribute to the synergic effects in relation to the concentrations of both substances. The presence of important enzymes in the cell membrane of H. pylori (such as urease) may contribute to the strong antibacterial effect of garlic and omeprazole against this micro-organism.
The MIC values for both raw garlic extract and the commercial garlic tablets were rather low, so that an in-vivo effect of garlic in the stomach seems possible. In-vivo studies have already shown the therapeutic potential of garlic in humans. 10 The gastric environment such as pH, temperature and dietary factors also influence the antibacterial activity of omeprazole and garlic compared with in-vitro conditions. The intragastric acidity is not expected to be a problem as omeprazole needs acid-catalysation for its antibacterial activity and an antibacterial effect of garlic was still found in the presence of a buffer with pH 2.
An antibacterial effect of garlic and a synergic effect of garlic in combination with omeprazole might be an interesting alternative therapy for H. pylori infections. Further studies concerning the combined effect of omeprazole and garlic are warranted.
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Notes |
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References |
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2 . McGowan, C. C., Cover, T. L. & Blaser, M. J. (1994).The proton pump inhibitor omeprazole inhibits acid survival of Helicobacter pylori by a urease-independent mechanism. Gastroenterology 107, 738 43.[ISI][Medline]
3 . Jonkers, D., Stobberingh, E. & Stockbrügger, R. (1996). Omeprazole inhibits growth of Gram-positive and Gram-negative bacteria including Helicobacter pylori in vitro. Journal of Antimicrobial Chemotherapy 37, 14550.[Abstract]
4 . Farbman, K. S., Barnett, E. D., Bolduc, G. R. & Klein, J.(1993). Antibacterial activity of garlic and onions: a historical perspective. Pediatric Infectious Disease Journal 12, 6134.[ISI][Medline]
5 . Feldberg, R. S., Chang, S. C., Kotik, A. N., Nadler, M., Neuwirth, Z., Sundstrom, D. C. et al. (1988).In vitro mechanism of inhibition of bacterial cell growth by allicin. Antimicrobial Agents and Chemotherapy 32, 1763 8.[ISI][Medline]
6 . Cellini, L., Di Campli, E., Masulli, M., Di Bartolomeo, S. & Allocati, N.(1996).Inhibition of Helicobacter pylori by garlic extract (Allium sativum ). FEMS Immunology and Medical Microbiology 13,273 7.[ISI][Medline]
7 . Sivam, G. P., Lampe, J. W., Ulness, B., Swanzy, S. R. & Potter, J. D.(1997).Helicobacter pylori in vitro susceptibility to garlic (Allium sativum ) extract. Nutrition and Cancer 27, 118 21.[ISI][Medline]
8 . Reuter, H. D., Koch, H. P. & Lawson, L. D. (1996). Therapeutic effects and applications of garlic and its preparations. In Garlic: the Science and Therapeutic Application of Allium sativum L. and Related Species (Koch, H. P. & Lawson, L. D., Eds), pp. 162- 72. Williams and Wilkins, Baltimore, MD.
9 . Rhodes, M. J. C. (1996).Physiologically-active compounds in plant foods: an overview. Proceedings of Nutrition Society 55, 371 84.[ISI]
10 . Hunan Medical College. (1980).Garlic in cryptococcal meningitis: a preliminary report of 21 cases. Chinese Medical Journal 93, 123.[ISI][Medline]
Received 26 October 1998; returned 29 December 1998; revised 10 February 1999; accepted 17 February 1999