Susceptibility of helicobacter pylori to essential oil of dittrichia viscosa subsp. revoluta

Phytother. Res. 22, 259–263 (2008)
SUSCEPTIBILITY OF HELICOBACTER PYLORI Published online 31 December 2007 in Wiley InterScience ( DOI: 10.1002/ptr.2284
Susceptibility of Helicobacter pylori to Essential
Oil of Dittrichia viscosa
subsp. revoluta
Graça Miguel1, Leonor Faleiro1*, Carlos Cavaleiro2, Lígia Salgueiro2 and Joseph Casanova3
1Universidade do Algarve, FERN, Campus de Gambelas, 8005-139 Faro, Portugal
2Faculdade de Farmácia, Laboratório de Farmacognosia/CEF, Universidade de Coimbra 3000 Coimbra, Portugal
3Université de Corse, UMR CNRS 6134, Equipe Chimie et Biomasse, Route des Sanguinaires, 20000 Ajaccio, France
The essential oil of Dittrichia viscosa subsp. revoluta and its fractions were assessed for anti-Helicobacter
activity. The essential oil was isolated by hydrodistillation, submitted to flash column chromatography and
analysed by gas chromatography, gas-chromatography coupled to mass spectrometry and 13C-nuclear magnetic
resonance. The anti-Helicobacter
activity was determined by incorporation of the crude essential oil and
oxygenated fractions of the oil into the culture medium. At a concentration of 0.025
μL/mL no recovery was
registered when one of the oxygenated fractions of the oil, mainly constituted by 3-methoxy cuminyl isobutyrate
(about 40%), was used. This fraction revealed a higher activity against the six H. pylori
strains tested when
compared with the other oxygenated fractions. The crude essential oil at a concentration of 0.33
reduced the initial population of H. pylori
CCUG 15818 of 8.52 ± 0.30 log cfu/mL to 7.67 ± 0.22 log cfu/mL.
The susceptibility of several Helicobacter pylori strains to the oxygenated fraction of Dittrichia viscosa subsp.
essential oil suggests the possible use of these natural products in combating this widespread infec-
tion. Copyright 2007 John Wiley & Sons, Ltd.

Keywords: Helicobacter pylori; Dittrichia viscosa; essential oil; antibacterial activity.
side effect profile (headache, dizziness, nausea, diarrhea, INTRODUCTION
pseudomembraneous colitis, mycosis, sore mouth andtongue, drug hypersensitivity and paraesthesia) are real Helicobacter pylori is a Gram-negative spiral-shaped problems of the triple therapy. The results of the double bacterium that colonizes the human stomach and duo- therapy on H. pylori eradication are also inconsistent denum, despite the relative inhospitable gastric environ- (Pakodi et al., 2000). Along with the development of ment to the majority of bacteria (Lamarque and Peek resistance to antibiotics there are other factors that Jr, 2003; Stoicov et al., 2004). Chronic gastric infection contribute to therapeutic failure: cost and efficacy of by H. pylori may originate various gastric-related antibiotics regarding the pH (for instance, amoxicillin diseases such as chronic gastritis, peptic ulceration and is most active at a neutral pH and tetracycline has gastric cancer. Most colonized individuals (approxi- greater activity at a low pH (Wang and Huang, 2005).
mately 80%) remain asymptomatic, presenting mild Many naturally occurring compounds found in the but diffuse inflammation of the stomach, showing little dietary and medicinal plants, herbs and fruit extracts or no atrophy throughout their lifetimes indicating have been reported to possess antimicrobial activities that the bacterium and the host adapt to each other (Lin et al., 2005; Li et al., 2005; Wang and Huang, 2005; through a long-term equilibrium (Blaser and Atherton, Chun et al., 2005). For instance, recent research has 2004; Joseph and Kirschner, 2004). On the other hand, demonstrated that resveratrol produced during wine there are those individuals that are chronically infected fermentation is active in acidic conditions (e.g. in stom- and develop severe disease such as adenocarcinoma ach) and may be linked to inhibition of H. pylori (Lin et al., 2005). Green tea catechins (epigallocatechin and A combination of therapeutic agents has been used epicatechin) have been shown to inhibit the growth in the eradication of H. pylori: (a) triple therapy with of H. pylori in a guinea-pig model of infection (Stoicov the antibiotics metronidazole, tetracycline or amoxicillin et al., 2004). In some cases, flavonoids and isoflavonoids and bismuth; (b) double therapy constituted by anti- present in ethanol extracts of some plants were revealed biotics jointly with proton-pump inhibitors (omeprazole) to possess potent anti-H. pylori activities (Fukai et al., or with H -blockers (ranitidine). The resistance to 2002; Li et al., 2005). Generally, flavonoids have a range metronidazole, in about 20% of patients especially of biological activities and pharmacological effects, those already treated with metronidazole, and the high including also a pronounced antiulcerogenic activity(Motilva et al., 1992). The activity of essential oils againstHelicobacter pylori was reported (Ohno et al., 2003) * Correspondence to: Leonor Faleiro, Universidade do Algarve, FERN, and amongst the 13 essential oils tested for H. pylori Campus de Gambelas, 8005-139 Faro, Portugal.
growth inhibition, Cymbopogon citratus (lemongrass) and Lippia citriodora (lemon verbena) were demon- Contract/grant sponsor: Fundação para a Ciência e a Tecnologia (FCT);contract/grant number: SFRH/BSAB/348/2003 (POCTI).
strated to have the highest bactericidal activities even Copyright 2007 John Wiley & Sons, Ltd.
Phytother. Res. 22, 259 – 263 (2008)
Copyright 2007 John Wiley & Sons, Ltd.
at low pH values (pH 4.0 and 5.0). During another i.d., film thickness 0.25 μm). Carrier gas: helium at investigation the combination of 0.0312 mg/mL of 0.8 mL/min; splitting ratio: 1:60; injection temperature: origanum and monolaurin was cidal for H. pylori strain 250 °C; oven temperature programmed from 60 to ATCC 49503 (Preuss et al., 2005).
220 °C at 2 °C/min and then held isothermal (20 min); Dittrichia viscosa W. Greuter Asteraceae is a subshrub widespread in Europe, especially in Mediterraneanareas (Devesa, 1987). This species has been used for Gas chromatography-mass spectrometry. Analyses were
years in folk medicine in the treatment of gastroduo- carried out in a Hewlett-Packard 6890 gas chromato- denal diseases (Font Quer, 1978). Some studies revealed graph fitted with a HP1 fused silica column (polydime- that the antiulcerogenic effect of D. viscosa is mainly thylsiloxane 30 m × 0.25 mm i.d., film thickness 0.20 μm), due to its flavonoid fraction (Grande et al., 1992; Martín interfaced with a Hewlett-Packard mass-selective detec- et al., 1988). In addition to the flavonoid fraction, other tor 5973 (Agilent Technologies) operated by HP Enhanced authors studied the volatile oil of D. viscosa collected ChemStation software, version A.03.00. GC parameters in Turkey and Spain (Pérez-Alonso et al., 1996; Camacho as described earlier; interface temperature: 250 °C; MS et al., 2000). The essential oil of D. viscosa subsp. viscosa source temperature: 230 °C; MS quadrupole tempera- collected in Portugal was also subjected to studies in ture: 150 °C; ionization energy: 70 eV; ionization current: order to elucidate its ability to prevent H. pylori growth 60 μA; scan range: 35 –350 units; scan/s: 4.51.
(Silva et al., 2005). The investigators concluded that thechemical composition of the oil isolated from plants 13C-NMR. 13C-NMR spectra of the crude oil as well as
collected in Portugal was closer to that previously the fractions of flash chromatography were recorded reported (Camacho et al., 2000) for the oil obtained on a Bruker AC200 Fourier Transform spectro- from plants from Spain (Province of Jaénz) and for meter operating at 50.323 MHz, equipped with a 10 mm that from Corsica (Blanc et al., 2006), mainly in the probe (200 mg oil; 2 mL CDCl ; 5000 scans) or 5 mm presence of the major components fokienol and (E)- probe (70 mg oil; 0.5 mL CDCl ; 10 000 scans), with all nerolidol. Moreover in that study, a small amount of shifts referred to tetramethylsilane (TMS). 13C spectra essential oil of D. viscosa subsp. viscosa from Portugal were recorded with the following parameters: pulse was detected, for the first time, which could drastically width (PW), 5 μs [or 3 μs] (flip angle 45°); acquisi- reduce the growth of H. pylori (Silva et al., 2005).
tion time, 1.3 s and relaxation delay (Dl), 2 s (total The main goal of the present contribution was recycling time, 3.3 s) for 32 K data table with a spec- to determine the ability of the essential oil of the tral width (SW) of 12 500 Hz (250 ppm); CPD mode Portuguese endemic D. viscosa subsp. revoluta and some decoupling; digital resolution, 0.763 Hz/pt. An exponen- of its fractions to inhibit the growth of H. pylori.
tial multiplication of the free induction decay with theline broadening of 1.0 Hz was applied before Fouriertransformation.
Qualitative and quantitative analyses. The identity of
the components was achieved from their retention
Plant material. The aerial parts of Dittrichia viscosa
indices on polar and apolar columns, determined subsp. revoluta were collected in the region of relative to the retention times of a series of C -C n- Algarve, Portugal in the flowering phase, during alkanes with linear interpolation with those of authen- tic components included in our own laboratory database.
Acquired mass spectra were compared with reference Isolation procedure. An essential oil sample was isolated
spectra from our own library or from literature data by water distillation for 4 h from fresh material, using a (Adams, 2004; Joulain and Konig, 1998). The identity Clevenger-type apparatus, according to the procedure was also determined by 13C-NMR spectroscopy, follow- described in the European Pharmacopoeia (Anonymous, ing the methodology developed and computerized in 1996). The essential oil was stored at 4 °C in the dark our laboratories (Tomi et al., 1995).
prior to analysis. The yield of the essential oil was 0.3% Relative amounts of individual components were calculated based on GC peak areas without flameionization detector (FID) response factor correction.
Oil fractionation. The bulk oil (2 g) was submitted
to flash chromatography (FC), silica gel 63–200 μm.
Antibacterial determination. Crude essential oil and
The first two fractions (F) were eluted with pentane oxygenated fractions of the oil were eluted in 2-propanol (F1 = 26 mg; F2 = 133 mg); two fractions were eluted (10%, v/v) and all concentrations were from the same with pentane/diethyl oxide (95/5) (F3 = 48 mg; F4 = stock solution of the eluted essential oil. Essential oil 134 mg); two fractions were eluted with pentane/diethyl and the oxygenated fractions of the oil were incor- oxide (75/25) (F5 = 459 mg; F6 = 443 mg); the last two porated into the Columbia agar medium (supple- fractions were eluted with diethyl oxide (F7 = 339 mg; mented with 10% blood, v/v). The 2-propanol was tested previously for antimicrobial activity and at theconcentration used no effect on bacterial viability was Gas chromatography. Analytical GC was carried out
registered. Helicobacter pylori strain 3, 28, 30, 40 and in a Perkin-Elmer Autosystem XL gas chromatograph 147 are clinical isolates from gastric biopsies obtained apparatus with dual FID and fused-silica capillary at Faro Hospital (Portugal) and belong to the Micro- columns with different stationary phases: BP-1 (poly- biology Laboratory of Faculty of Natural Resources methylsiloxane 50 m × 0.22 mm i.d., film thickness Engineering of University of Algarve. H. pylori strains 0.25 μm), and BP-20 (polyethylene glycol 50 m × 0.22 mm CCUG 15818, 26695 and J99 were used as laboratory Copyright 2007 John Wiley & Sons, Ltd.
Phytother. Res. 22, 259 – 263 (2008)
SUSCEPTIBILITY OF HELICOBACTER PYLORI TO ESSENTIAL OIL strains. Bacterial viability was determined by the dropmethod (Chen et al., 2003). Briefly, the dilutions of the RESULTS AND DISCUSSION
sample were done using a 96-well plate, initially 250 μLof the sample was distributed into the first well of each The identified compounds in the essential oil of D. row of the microplate followed by the preparation of viscosa subsp. revoluta are indicated in Table 1, where 10-fold serial dilutions using a multichannel pipette the components are listed in order of their elution (Transferpette-8, Brandtech, USA) by transferring 20 μL on the BP-1 column. The essential oil of D. viscosa from the first row into 180 μL of medium on the next subsp. revoluta was mainly constituted by oxygenated column. The inocula were homogenized by pipetting compounds. The fractions eluted with pentane/diethyl at least 10 times and the followed dilutions were done oxide (75/25), that is, fraction F5 and F6 provided 459 by repeating the process having changed pipette tips and 443 mg, respectively; the elution with diethyl oxide between dilutions. Afterwards, six replicates of 10 μL produced fractions F7 and F8 with a total mass of 339 from each of the six selected dilutions were distributed and 88 mg, respectively. The major components present onto Columbia agar medium (supplemented with 10% in the oil were 3-methoxy cuminyl isobutyrate (12%), blood, v/v) at appropriate essential oil concentration.
α-cadinol (6.3%), eudesm-6-en-4α-ol (4.8%) and δ- Inocula were allowed to dry before to introduce them cadinene (4.6%). The presence of 3-methoxy cuminyl in anaerobic jars (Oxoid, Basingstoke, Hampshire, UK) isobutyrate was previously reported by Ascensão et al. in the presence of Anaerocult A pack for generation (1999) in the oil of D. viscosa subsp. revoluta repre- of anaerobic conditions. The plates were incubated senting 15% of the total oil of the inflorescences. The presence of costic acid, isocostic acid and 4-en-ilicic The MIC of the antibiotic amoxicillin was determined acid were reported by Blanc et al. (2006, 2005, 2004) by the ε-test (AB Biodisk, Sweden) in Columbia medium for D. viscosa ssp. viscosa and Inula graveolens from Corsica. The direct quantification of these three Table 1. Composition of the essential oil of D. viscosa subsp. revoluta. Components listed according to their
elution on the BP1 column

a RI: retention indices measured on apolar column (BP-1).
b RI: retention indices measured on polar column (BP-20).
nd: not determined.
Copyright 2007 John Wiley & Sons, Ltd.
Phytother. Res. 22, 259 – 263 (2008)
eudesman-type acids by GC was not performed due totheir relative low volatility, being necessary to proceedto a previous derivatization in order to transform themin the corresponding methyl esters prior to the GCanalysis (Grande et al., 1992; Blanc et al., 2005).
Regarding this, such acids were identified by 13C-NMR.
The fractions used for the determination of anti-H.
activity were those mainly composed of oxygen-ated compounds not only by their highest mass ob-tained but also by the oxygenated components that aregenerally responsible for biological activities, namelyfraction 4, 5, 6 and 7. 3-Methoxy cuminyl isobutyratewas the major component detected in fraction F5constituting about 40% of the fraction. In fraction 6,one component not identified constituted 25% ofthe total oil, followed by T-cadinol, T-muurolol andcaryophylla-4(14),8(15)-diene-5α-ol that reached apercentage of 18% and finally eudesm-6-en-4α-ol (10%).
The fraction F7 was mainly constituted by α-cadinoland α-eudesmol with a total percentage of 31%. Theolefinic fractions F1 and F2 eluted by pentane weremainly composed of α-copaene (34%), γ-cadinene (19%), α-ylangene (15%), α-gurjunene (14%), α-muurolene(10%) and δ-cadinene (10%).
Crude essential oil of Dittrichia viscosa subsp. revoluta demonstrated to have anti-Helicobacter activity deter- Figure 1. Susceptibility of Helicobacter pylori strains CCUG
mined against the laboratory strain CCUG 15 818. At 15 818, 26 695, J99, 3, 28 and 40 to the oxygenated fractions a concentration of 0.33 μL/mL the initial population 5 (A) and 7 (B) of the essential oil of Dittrichia viscosa subsp.
of 8.52 ± 0.30 log cfu/mL was reduced to 7.67 ± revoluta. Data are the mean of three independent experiments.
Bars represent the standard deviation.
Susceptibility of H. pylori strain CCUG 15 818 to all oxygenated fractions tested; F4, F5, F6 and F7 wasobserved. However, fraction 5, mainly constituted of3-methoxy cuminyl isobutyrate, showed the highest that are highly resistant to the antibiotic amoxicillin activity against the seven strains used, followed by frac- are susceptible to oxygenated fraction F5.
tion F7 mainly composed of α-cadinol and α-eudesmol.
In spite of chemical differences detected in both Anti-Helicobacter activity of the two active fractions is subspecies, our previous results on the determina- represented in Fig. 1. H. pylori strain CCUG 15 818 tion of the antimicrobial activity of Dittrichia viscosa was the most susceptible; at a concentration of 0.025 μL/ subsp. viscosa essential oil indicated a specific activity mL no growth was registered. H. pylori strains J99 and of this essential oil against H. pylori but a null activity strain 3 were middle resistant. At a concentration of against an important foodborne pathogen Listeria 0.03 μL/mL the initial population of strains J99 (9.61 ± monocytogenes (Silva et al., 2005). The use of the 0.07 cfu/mL) and strain 3 (9.77 ± 0.28 log cfu/mL) were essential oil of Dittrichia viscosa subsp. viscosa and reduced 1 log. At the highest concentration no recov- D. viscosa subsp. revoluta may greatly contribute to an ery of cells was registered. The most resistant strains efficient control of this bacterial pathogen that is spread were strain 26 695 and strain 147. At the highest con- amongst the world population and for which the mode centration tested (0.035 μL/mL) the initial population of transmission is still controversial. Moreover due to of strain 26 695 (9.02 ± 0.76 log cfu/mL) was reduced the increase of the multiresistance pattern and also the 4 log, whereas the initial population of strain 147 increasing tendency of the public to consume ‘green (9.04 ± 0.07 log cfu/mL) was reduced 6 log. Fraction 7 products’ the use of these types of compounds will demonstrated an anti-Helicobacter pylori activity only greatly help to combat this infection accompanied against the strain H. pylori CCUG 15 818 (Fig. 1). At by consumer confidence and support which plays an a concentration of 0.025 μL/mL the initial population important role to the therapy success.
of this strain (9.01 ± 0.04 log cfu/mL) was reduced 5 log.
The other strains did not experience a significant re-duction (Fig. 1). Regarding the susceptibility of H. pylori Acknowledgement
strains to the antibiotic amoxicillin the MIC value forH. pylori strain J99, CCUG 15 818, 147, was 0.50 μg/ We thank Dr Maria de Lurdes Monteiro from Instituto Nacional de mL, 0.16 μg/mL, 0.16 μg/mL and H. pylori strains 3, Saúde, Dr Ricardo Jorge (Lisboa, Portugal) for providing the strainsof Helicobacter pylori CCUG 15 818, 26 695 and J99. We are grateful 28 and 40 were resistant to higher than 256 μg/mL. It is to the Fundação para a Ciência e a Tecnologia (FCT) for a grant to interesting to verify that the H. pylori strains 3 and 28 M. G. Miguel SFRH/BSAB/348/2003 (POCTI).
Copyright 2007 John Wiley & Sons, Ltd.
Phytother. Res. 22, 259 – 263 (2008)
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