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Volume 8 • Number 3 • 2003 H E L I C O B AC T E R

Antimicrobial Activity of Essential Oils against Helicobacter pylori Blackwell Publishing Ltd.

Tomoyuki Ohno,* Masakazu Kita,† Yoshio Yamaoka,*,‡ Shigeyoshi Imamura,* Toshiro Yamamoto,† Shoji Mitsufuji,* Tadashi Kodama,* Kei Kashima* and Jiro Imanishi† *Third

Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, 602–8566, Japan; †Department of Microbiology, Kyoto Prefectural University of Medicine, Kyoto, 602–8566, Japan; and ‡Department of Medicine,Veterans Affairs Medical Center and Baylor College of Medicine, Houston, TX 77030, USA ABSTRACT

Background. Helicobacter pylori is an important pathogen responsible for gastroduodenal diseases in humans. Although the eradication of H. pylori using antibiotics often improves gastroduodenal diseases, resistance to the antibiotics is emerging. Materials and Methods. The antimicrobial effect of essential oils and the development of resistance to the essential oils were evaluated in vitro and in vivo. Results. Thirteen essential oils used in this study completely inhibited the growth of H. pylori in vitro at a concentration of 0.1% (v/v). Cymbopogon citratus (lemongrass) and Lippia citriodora (lemon verbena)

were bactericidal against H. pylori at 0.01% at pH 4.0 and 5.0. Resistance to lemongrass did not develop even after 10 sequential passages, whereas resistance to clarithromycin developed under the same conditions. In in vivo studies, the density of H. pylori in the stomach of mice treated with lemongrass was significantly reduced compared with untreated mice. Conclusions. These results demonstrate that the essential oils are bactericidal against H. pylori without the development of acquired resistance, suggesting that essential oils may have potential as new and safe agents for inclusion in anti-H. pylori regimens.


also been used for many years to treat a variety of medical ailments, although the mechanism of their action is generally not clarified [17]. Recently, the antimicrobial activity of natural compounds has been reported [18,19], including a few reports on the effects of essential oils against H. pylori [20,21], however, no animal experimentation was reported. In the present study, we investigated the antibacterial activity of essential oils against H. pylori both in vitro and in vivo. We also investigated the development of H. pylori resistance to the essential oils in vitro.

elicobacter pylori is now recognized as an important causal agent in gastroduodenal diseases, such as chronic gastritis, peptic ulceration, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (MALToma) [1–6]. Infection can be cured in > 80% of H. pyloriinfected patients using standard therapies [7,8]. However, emerging resistance of H. pylori to antibiotics, especially metronidazole and clarithromycin, limits the use of these antibiotics in the treatment of H. pylori infection [9–12]. Antimicrobial substances other than antibiotics would be very useful in the treatment of H. pylori infection if they were shown to be effective against both antibiotic-resistant and -susceptible H. pylori strains. In fact, a number of drugs and natural substance such as ecabet sodium, tea cathechins, garlic extracts and honey have been shown to have an antibacterial effect against H. pylori in vitro [13–16]. Essential oils, which are extracted from plants (e.g. leaves, peels) have

Materials and Methods

H. pylori Strains Seven H. pylori strains were used in this study. ATCC43504 was obtained from the American Type Culture Collection (Rockville, MD, USA). Strains KP142B, KP143B, KP146B, KP206B and KP223B were isolated from human gastric biopsy specimens at Kyoto Prefectural University of Medicine. CPY2052 was a kind gift

Reprint requests to: Tomoyuki Ohno MD, Mailing address: Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, 465 Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602–8566, Japan. © 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215


208 from Dr M. Karita (Houfu Onsen Hospital, Yamaguchi, Japan). All H. pylori strains used in this study were cag pathogenicity island positive and was of the vacA s1-m1 genotype according to our previous study [22]. Essential Oils We used 13 essential oils [Cupressus sempervirens (cypress), Juniperus communis (juniper), Melaleuca alternifolia (tea tree), Lippia citriodora (lemon verbena), Ocimum basilicum album (tropical basil), Mentha piperita (peppermint), Origanum marjorana (marjoram sweet), Eucalyptus globulus (eucalyptus), Ravensara aromatica (ravensara), Lavandula latifolia (lavender), Citrus limonum (lemon), Cymbopogon citratus (lemongrass), and Rosmarinus officinalis (rosemary camphor)]. All the essential oils were kindly supplied by Laboratoire Sanoflore, Ltd (Lozeron, France), and data on the gas liquid chromatograph analysis were provided [Table 1]. The Antibacterial Activity of Antibiotics Minimum inhibitory concentrations (MICs) were determined using the agar dilution method with slight modefication [23]. Briefly, approximately 1 × 106 colony forming units (CFU) of H. pylori strains were plated onto brain heart infusion (BHI) (Nissui Chemical Co., Tokyo, Japan) agar containing from 64 to 1.0 µg/ml of antibiotics (amoxicillin, clarithromycin, metronidazole or tetracycline). After incubation for 5 days at 37°C with an Aeropack system (Mitsubishi Gas Chemical Co. Ltd, Tokyo, Japan), colonies were counted. MICs were determined as the lowest concentration of antibiotics inhibiting visible growth.

Ohno et al. approximately 1 × 107 CFU of H. pylori. The test tubes were incubated for 48 hours under a gas mixture consisting of 80% N2, 15% CO2 and 5% O2 on a reciprocal shaker (200 r.p.m.). Following incubation, 100 µl aliquots of the medium were harvested and plated onto blood agar plates (Nissui Chemical). Emergent colonies were counted after incubation for 5 days, the same as above. Time Course A suspension of H. pylori ATCC43504 (∼1 × 107 CFU) was inoculated into 10 ml of culture medium with DMSO (0.2% [v/v]) containing 0–0.02% (v/v) of essential oils and incubated for 48 hours at 37°C under microaerophilic conditions. Samples (100 µl) were taken at 0, 6, 12, 24 and 48 hours after inoculation for viable cell counting. Colonies were counted in the same manner as described above. Bactericidal Activity of the Essential Oils at Various pHs The effects of pH on the antibacterial activities of essential oils were assessed by using methods reported previously with slight modification [16]. Briefly, the pH of the buffer was adjusted to 4 or 5 using either 100 mM citrate buffer (pH 5.2), or to pH 6 or 7 using 10 mM phosphate buffer (pH 6.8). H. pylori ATCC43504 (∼1 × 107 CFU) was inoculated into 10 ml of the medium with DMSO (0.2% [v/v]) to which various concentrations of the essential oil were added (from 0.0025 to 0.1% [v/v]). All tubes were incubated at 37°C. Twenty µl of sample were taken at 0, 15, 30 and 60 minutes after inoculation to enumerate the number of viable cells. Test for Development of Resistance to Essential Oils

The Antibacterial Activity of Essential Oils As there is no established method to determine the antibacterial activity of essential oils against H. pylori strains, we used the broth microdilution methods according to a previous report [18] with a slight modification. Briefly, BHI broth (including control) was supplemented with 0.2% (v/v) of dimethyl sulfoxide (DMSO) (Wako Chemicals Co., Tokyo, Japan) to enhance oil solubility. We confirmed that DMSO had no bactericidal effect against H. pylori at this concentration (data not shown). Broth containing 0, 0.1 and 0.01% (v/v) of essential oils was added to 10 ml of diluted culture medium containing

To investigate whether resistance developed, four strains (ATCC43504, CPY2052, KP142B, and KP143B), which were sensitive to clarithromycin were used (MIC ≤ 1.0 µg/ml). Agar dilution assays were performed by adding lemongrass oil in serial two-fold dilution from 0.0025% to 0.64% (v/v), or clarithromycin from 0.01 to 8 µg/ml to BHI agar with or without a test substance. H. pylori (1 × 107 CFU) was inoculated onto the plates and incubated for 5 days. After determination of the MIC by the plate colony counting technique, colonies surviving at the maximum concentration were passaged 10 times on the same type of plates. © 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215

Compound (common name) 1 Limonene 2 Myrcene 3 Citronellal 4 Linalool 5 Neral 6 Geranial 7 Geranyl acetate 8 α-Pinene 9 Sabinene 10 1,8-Cineol 11 trans-β-Ocimene 12 β-Caryophyllene 13 Germacrene D 14 Farnesene 15 Camphene 16 β-Pinene 17 para-Cymene 18 Campher 19 bornyl Acetate 20 α-Terpineol 21 Borneol 22 Verbenone 23 γ-Terpinene 24 Terpinolene 25 Terpinene-4-ol 26 α-Terpinene 27 Aromadendrene 28 δ-3-Carene 29 Terpenyl acetate 30 Cadinene 31 Cedrol 32 β-Phellandrene 33 γ-Terpinene 34 γ-Elemene

Cymbopogon citratus album (Lemongrass)

Lippia citriodors (Lemon Verveine)

Rosmarinus officinalis (Rosemary Camphor)

6.7 0.5 0.51 1.92 37.38 37.5 0.74


3.73 3.89

Citrus limonum (Lemon)

Melaleuca alternifolia (Tea Tree)



Cupressus Juniperus sempervirens communis (Cypress) ( Juniper) 3.98 2.4

Ocimum basilicum album (Tropical Basil)

5.05 4.35

Mentha piperita (Peppermint) 1.58

0.95 12.04 18.46 1.32 0.89 2.48 2.36 3.52 6.31 4.04 5.66


3.08 0.37 3.22


62.16 0.52



1.13 3.62

8.2 0.9


1.76 1.63

1.7 2



Eucalyptus globulus (Eucaryptus Globulus)

Ravensara aromatica (Ravensara)

Lavendula latifolia (Lavender)

3.9 1.35




0.76 4.64

0.9 9.83 2.97 1.77 15.61 1.25 2.04 3.2 0.68

31.05 16.5

Origanum marjorana (Majoram sweet)

21.45 14.15 1.8



Essential Oils and H. pylori

© 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215

Table 1 Chemical and percentage(%) composition of the essential oils

5.42 12.79 57.04 3.07

2.74 4.9

3.35 13.9

1.15 0.46

0.39 2.21 8.85

23.39 3.72 34.15 10.85 1.68

0.58 2.43 0.87

2.5 0.15 0.1





26.15 2.65


2.05 1.8

15.72 1.53 0.73 1.36 4 5.4 2.4



Table 1 continued

Compound (common name)

Lippia citriodors (Lemon Verveine)

Rosmarinus officinalis (Rosemary Camphor)

Citrus limonum (Lemon)

Melaleuca alternifolia (Tea Tree)

Cupressus Juniperus sempervirens communis (Cypress) ( Juniper)

Ocimum basilicum album (Tropical Basil)

Mentha piperita (Peppermint)

Origanum marjorana (Majoram sweet)

Eucalyptus globulus (Eucaryptus Globulus)

Ravensara aromatica (Ravensara)

Lavendula latifolia (Lavender)

1.05 3.2 83.5 2.2 1.3


21.79 3.8 3.18 4.87 3.21 38.25 2.06 2.9 6.55 3.3

35.09 1.6 0.8 0.52 1.5 3.49 1.51 2.67

Ohno et al.

© 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215

35 δ-Cadinene 36 Limonene + 1,8-Cineol 37 Methyl Chavicol 38 Eugenol methyl Ether 39 trans-α-Bergamotene 40 Menthone 41 Menthofuran 42 Isomenthone 43 Menthyl acetate 44 Neomenthol 45 Menthol 46 Pulegone 47 trans-thuyanol 4 48 cis-thuyanol 4 49 Linalyl acetate 50 trans-Pinocarveol 51 Globulol 52 Octene1-ol-3 53 Octanone-3 54 cis-β-Ocimene 55 Lavendulol 56 Lavendulyl Acetate

Cymbopogon citratus album (Lemongrass)


Essential Oils and H. pylori In Vivo Studies We used the mouse model of H. pylori infection previously established in our laboratory [24]. In this model, H. pylori CPY2052 continued to colonize the stomachs of mice for 15 months after inoculation. Four-week-old, specific-pathogenfree, male ICR mice (Charles River Japan, Atsugi, Japan) were maintained on sterile food and water. Mice were orally inoculated on three consecutive days with freshly prepared CPY2052, approximately 3 × 108 CFU in 0.5 ml of BHI broth. Two weeks later, the mice were divided into three groups. The first group received 0.5 ml lemongrass oil (0.1% [v/v]) dissolved in distilled water with DMSO (0.2% [v/v]); the second group received only 0.5 ml distilled water with DMSO (0.2% [v/v]) by oro-gastric tube for 14 days, and the third group received nothing as control. As lemongrass oil should be diluted in the gastric juice of the mouse’s stomach, we decided to choose the concentration of lemongrass oil as 0.1%, which was higher than MIC in vitro (see Results section). After 2 weeks of treatment, the mice were sacrificed under anesthesia. The entire stomach was resected and homogenized in 1 ml phosphate buffer saline. A 100-µl aliquot from each stomach homogenate was spread onto BHI agar plates containing 7% (v/v) horse blood, vancomycin (10 µg/ml), polymyxin B (2.5 U/ml), trimethoprim (5 µg/ml) and amphotericin B (2 µg/mL) (Helicobacter selective BHI agar plate) (Eiken Chemical Co., Tokyo, Japan) and incubated for 5 days at 37°C. The colonies were then counted blindly without the information of the mouse group. All protocols involving animal experimentation were approved by the Animal Care and Ethics Committee at the Kyoto Prefectural University of Medicine. Statistical Analysis Colonization was expressed as median ± standard deviation. Kruskal–Wallis test were used for statistical analysis. A p-value of < 0.01 was considered significant.

antibiotics (amoxicillin, clarithromycin, metronidazole and tetracycline) used in this study. Clinical isolates from our laboratory were resistant to one or more of the antibiotics. Strains KP142B, KP143B and KP206B were resistant to metronidazole (MIC: 4, 2 and 64 µg/ml, respectively). Strain KP146B was resistant to metronidazole (MIC: 2 µg/ml) and clarithromycin (16 µg/ml), and strain KP223 was resistant to metronidazole (8 µg/ml) and tetracycline (2 µg/ml). The in vitro antimicrobial effect of the 13 essential oils demonstrated that all essential oils completely inhibited the growth of both H. pylori strains at a concentration of 0.1% (v/v). Lemongrass inhibited the growth of all H. pylori strains studied (either antibiotics-susceptible or -resistant strains), even at a concentration of 0.01% (v/v). Lemon verveine inhibited the growth of five of seven H. pylori strains (except strains KP146B and KP223B) at a concentration of 0.01% (v/v). The other essential oils showed no bactericidal effect at this concentration (data not shown). Time Course Because lemongrass and lemon verbena had strong bactericidal effects against H. pylori, we examined the killing curve time course of these two oils. At a concentration of 0.02% (v/v), both oils killed strain ATCC43504 completely after 12 hours of incubation. At a concentration of 0.01% (v/v), only lemongrass killed strain ATCC43504 completely after 48 hours of incubation [Fig. 1]. Effect of the Essential Oils on the Growth of H. pylori in Buffers at Various pHs The viability of strain ATCC43504 at various pHs was investigated using lemongrass at concentrations from 0 to 0.02% (v/v). As shown in Fig. 2, lemongrass showed a concentration-dependent bactericidal effect at pH 4.0 and 5.0 but not at pH 6.0 or 7.0. At pH 4.0, visible colonies of strain ATCC43504 were significantly reduced after 60 minutes of incubation. Lemon verbena also showed bactericidal activity against H. pylori in much the same manner as demonstrated by the lemongrass (data not shown).


Antibacterial Activity Against H. pylori

Test for Resistance to Lemongrass

MICs of antibiotics for H. pylori were determined. Strains ATCC43504 and CPY2052 were susceptible (MIC < 1 µg/ml) to the four

We investigated whether H. pylori would acquire resistance to lemongrass. As shown in Fig. 3, all the H. pylori strains (strains ATCC43504, CPY2052,

© 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215


Ohno et al.

Figure 2 Effects of lemongrass on the growth of H. pylori at various pHs. H. pylori ATCC43504 were exposed to lemongrass at concentrations of 0.02 (), 0.01 (), 0.005 (), 0.0025 () and 0 ()% (v/v) for 60 minutes. Samples were taken at the time indicated, and the colonies developed were counted by the plate colony counting.

Figure 1 Effect of the essential oils on the growth of H. pylori in liquid medium. H. pylori ATCC43504 was cultured in BHI broth under microaerophilic conditions at 37°C with reciprocation for 48 hours. H. pylori ATCC43504 was exposed to lemon verbena at concentrations of 0.02 (), 0.01 (), 0.005 () and 0.0025 ()% (v/v), and to lemongrass at 0.02 (), 0.01 (), 0.005 () and 0.0025 ()% (v/v). Culture samples were taken at the time indicated, and the colonies developed were counted by the plate colony counting technique.

effect against H. pylori in vitro. As shown in Fig. 4, one of the 10 mice (10%) in the group that received the lemongrass oil was completely cured. H. pylori colonies recovered from the stomachs of mice treated with lemongrass were significantly reduced compared with mice given distilled water and mice without treatment (the median of colonies was 2.45 × 104 CFU/stomach for lemongrass [excluded one mouse in which the bacteria had been eradicated]; 1.45 × 105 CFU/stomach for distilled water, and 1.82 × 105 CFU/stomach for nontreated). Discussions

KP142B, and KP143B) acquired resistance to clarithromycin at MICs ranging from 4 to 32 µg/ml after 10 sequential passages. In contrast, no resistance was acquired for lemongrass oil (MIC range from 0.005 to 0.02% [v/v]) even after 10 passages. In vivo Studies We examined bactericidal effect of lemongrass in vivo because lemongrass had the most bactericidal

Because the prevalence of antibiotic-resistant H. pylori strains is increasing, the search for safe and effective nonantibiotic agents is essential. The traditional use and anecdotal evidence of plants as medicine provide the basis for suggesting that essential oils and plant extracts may be useful for specific medical conditions. In this study, we showed that essential oils, especially lemongrass and lemon verbena had bactericidal effects against H. pylori. © 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215

Essential Oils and H. pylori

Figure 3 Development of resistance to clarithromycin and lemongrass in vitro. H. pylori strains ATCC43504 (), CPY2052 (), KP142B (), and KP143B (), were exposed to various concentrations of clarithromycin (broken line, g/ml) and lemongrass (solid line,% [v/v]). After determination of the MIC by the plate colony counting technique, colonies present at the maximum concentration were passaged 10 times on the same type of plates.

Studies indicate that essential oils have bactericidal effects against several bacteria [19–21]. However, there are differences in the bactericidal activity among essential oils, although the exact reasons have not yet been clarified. Several components of essential oils, which are different in each essential oil, have the ability to disrupt or penetrate lipid structure [25]. Geranial (included at 37.5% in lemongrass and at 18.6% in lemon verbena) and neral (at 37.4% in lemongrass and at 12.0% in lemon verbena) may possess heightened anti-H. pylori bactericidal effects compared with other components, because geranial and neral were not found in the other 11 essential oils used in this study [Table 1]. This hypothesis is in agreement with previous studies that have reported these components to have antimicrobial and antifungal properties [26]. It will be necessary to examine each antibacterial component of essential oil separately, and in combination to ascertain whether they act alone or synergistically. H. pylori resides in the stomach and can resist pH levels lower than 3.0 [27]. In our studies, H. pylori strains can survive at pH 4.0, and the bactericidal effects of the essential oils were stronger at low pH. Several agents that have bactericidal effects against H. pylori are dependent on pH. For example, tea catechin demonstrates a strong H. pylori bactericidal effect at high pH © 2003 Blackwell Publishing Ltd, Helicobacter, 8, 207–215


Figure 4 Effect of lemongrass on H. pylori colonization in vivo. The bacterial counts of each group (excluding the mouse in which the bacteria had been eradicated-*) were shown. The end of the box indicate the 25th and 75th percentiles, a line inside it marks the median. Bars indicate the 10th and 90th percentiles, and the circles mark all data.

[13], whereas ecabet sodium is more effective at low pH [16]. Ecabet sodium is thought to bind to the cell wall and the level of binding is higher at low pH. It has been suggested that essential oils may show antibacterial activity in the stomach because they are more effective at lower pH. In this study, H. pylori strains resistant to clarithromycin emerged after 10 in vitro passages, whereas no strains resistant to the essential oils emerged after 10 passages in vitro. In addition, the essential oils had equivalent bactericidal effects against both antibiotic-susceptible strains (ATCC43504 and CPY2052), and other H. pylori strains which have resistance to antibiotics. These findings suggest that essential oils may be prime candidates as agents to treat H. pylori infection. Furthermore, the essential oils showed some antimicrobial effects in vivo. Lemongrass decreased the number of colonies in the mouse stomach ( p < 0.01). H. pylori was completely eradicated in one of 10 mice. Previous studies reported that colony density were associated with peptic ulcer and gastric inflammation [28– 30]. These facts indicate that essential oils could be used in combination, as even antibiotics with high activity are not effective when used singly. Additional studies are necessary to determine the correct dose and dosing interval of the

214 essential oils and to devise a drug delivery system which will prolong the gastric-transit time. In conclusion, the essential oils, especially lemongrass and lemon verbena, have strong bactericidal effects on the growth of H. pylori of both antibiotic-susceptible and -resistant strains even under acidic conditions. Moreover, no H. pylori strains resistant to essential oil emerged. In vivo mouse model showed that the colonies were reduced by oral administration of lemongrass. Lemongrass and lemon verbena may have potential as new and safely acting therapeutic regimen for H. pylori infection. We thank Dr Michael. S. Osato and Dr David Y. Graham (Baylor College of Medicine, Houston, TX) for critical reading of our manuscript. We also thank Hyperplants Co. Ltd, Tokyo, Japan for a gift of the essential oils. References 1 Graham DY. Helicobacter pylori infection in the pathogenesis of duodenal ulcer and gastric cancer: a model. Gastroenterology 1997;113:1983–91. 2 International Agency for Research on Cancer. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 61 Schistosomes, Liver Flukes and Helicobacter pylori. Lyon, France: World Health Organization, 1994. 3 Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984;1:1311–5. 4 Nomura A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med 1991;325:1127–31. 5 Raus EA, Langenberg W, Houthoff HJ, Zanen HC, Tytgut GN. Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the effects of antibacterial and antiulcer treatment. Gastroenterology 1987;94:33– 40. 6 Wotherspoon AC, Doglioni C, Diss TC, et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet 1993;342:575–7. 7 Axon A. Eradication of Helicobacter pylori: omeprazole in combination with antibiotics. Scand J Gastroenterol 1993;31:82–9. 8 Misiewicz J, Harris AW, Bardhan KD, et al. One week triple therapy for Helicobacter pylori: a multicentre comparative study. Gut 1996;41:735– 9.

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Antimicrobial activity of essential oils against helicobacter pylori