Abstract
Helicobacter pylori is the primary etiologic agent of peptic ulcer, duodenal ulcer, chronic gastritis, gastric adenocarcinoma and related gastroduodenal disorders. Current triple therapy, including antibiotics and proton-pump inhibitors, has been successful; however, adverse events, non-patient compliance and consequent relapse of Helicobacter pylori infections are common. Crude methanol extracts of Eucalyptus grandis Hill ex. Maiden (Myrtaceae) stem bark were screened against a standard strain ATCC 43504 and ten clinical strains of H. pylori using the agar diffusion method on Mueller-Hinton agar supplemented with defibrinated horse blood and grown in a microaerophilic incubator. All the strains except UCH 97002 and UCH 98020 were inhibited by the extract to varying degrees. The minimum inhibitory concentration (MIC) against the susceptible strains tested ranged from 0.39 and 1.56 μg/mL. The urease activity of the three H. pylori strains tested decreased with increasing concentrations of the extract. The greatest inhibition of urease activity was observed in clinical strain UCH 97009. In addition, methanol extracts of the E. grandis enhanced cell aggregation of seven of the H. pylori strains leading to a decrease in the cell surface hydrophobicity. The salt aggregation test titer decreased from >3 to <1.5 for five of the strains and to <3 for two of the strains. Phytochemical screening of the plant revealed the presence of tannins, essential oils and saponins, while alkaloids were not detected. The anti-Helicobacter pylori activity observed in this study correlates well with the traditional use of this plant in Nigeria.
Introduction
Helicobacter pylori is a Gram-negative spiral-shaped, fastidious, microaerophilic bacillus which rapidly hydrolyses urea as part of its adapted survival methods (CitationGoodwin et al., 1986). It has been implicated as the etiologic agent of gastritis, peptic ulcer, duodenal ulcer, gastric adenocarcinoma and related gastroduodenal disorders (CitationBuck, 1990). The bacterium produces high levels of urease, which it uses to hydrolyse urea, thereby releasing ammonia, which neutralizes acid in the gastric mucosa allowing survival of the bacterium and initial colonization (CitationEston et al., 1991; CitationNagata et al., 1993). Successful treatment of chronic H. pylori infections leads to the resolution of gastritis and diminished ulcer recurrence (CitationNIH, 1994). Unfortunately, eradication of H. pylori has proved to be difficult, and the optimal regimen has not been defined. Triple therapy appears to be most effective (CitationGlupczynski & Burette, 1990; CitationMarshall, 1993), with combinations of bismuth and metronidazole with either tetracycline or amoxicillin eliminating infection in 73% to 94% of cases. However, this treatment regimen produces serious adverse events in up to 30% of patients leading to their non-compliance and consequent relapse (CitationDavid, 1996).
Thus, the search for novel antimicrobial agents to eradicate H. pylori and yield better therapeutic results is of critical importance, especially in developing countries where the rates of H. pylori infections are high. To this end we have been investigating Nigerian medicinal plants based on their ethnomedical use to treat gastrointestinal diseases or having antimicrobial activities. One such plant is Eucalyptus grandis Hill ex. Maiden (Myrtaceae). This tree is native to Australia, and is one of the introduced species of Eucalyptus in Nigeria, where it has numerous ethnomedical applications. The genus Eucalyptus is used to treat sore throats and bacterial infections of respiratory, gastrointestinal and urinary tracts (CitationOlaniyi, 1982). An essential oil is applied or rubbed over the chest and throat to relieve catarrh and feverish conditions and the leaves are chewed for bad breath (CitationGill, 1992). Groups of chemical constituents have been reported including tannins, glycosides, flavonoids, esters and terpenes in the genus (CitationOyedeji et al., 1999). In terms of E. grandis, the leaves contain 0.12-0.26% of α-pinene, as well as the phenolic compound grandinol (CitationGlasby, 1991). In addition, flavesone, leptospermone, isolelptosepermone, grandinol, homo-grandinol and eugenol have also been isolated from the plant (CitationGhisalberti, 1996). The aim of his work is to investigate the antimicrobial effect of the methanol extract of the stem bark of E. grandis on H. pylori with reference to susceptibility, effect on urease activity and cell surface hydrophobicity.
Materials and methods
Plant collection and authentication
The stem bark of Eucalyptus grandis was collected in March 2000 from Jericho and Iwo Road, Ibadan, Nigeria, with the assistance of Felix Usang of the Forest Research Institute of Nigeria (FRIN). It was authenticated in the FRIN herbarium by Gabriel Ibahanesaboh. A voucher specimen was deposited at FRIN with the herbarium number (FHI 106044). The stem bark was air-dried and ground to a coarse powder.
Plant extraction and preparation of extracts
Coarsely powdered stem bark (50 g) was exhaustively extracted using a Soxhlet extractor with hexane and methanol as solvents for 24 h in succession. Each extract was filtered, concentrated under reduced pressure, and stored at 40°C until needed for analysis. Dilution of the dried extract to the final concentrations of 6.5, 25, and 50 μg/mL were made in 50% v/v methanol, respectively. Tween 80 was added to enhance proper dissolution of the extracts and the solutions were then used for the various assays.
Strains of Helicobacter pylori and culture methods
Eleven strains of H. pylori, a standard ATCC 43504, and ten clinical isolates, were used for this investigation. All strains were cultured from gastric biopsy specimens of patients attending the endoscopy unit of University College Hospital (UCH) Ibadan, Nigeria. H. pylori cells were identified according to colony morphology, Gram staining, microaerophilic growth (at 37°C), oxidase, catalase, urease, nitrate, H2S, hippurate hydrolysis- and nalidixic acid. The strains were coded as UCH97001, UCH97002, UCH97009, UCH98020, UCH98026, UCH99039, UCH99041, UCH99045, UCH99050, and UCH99052.
The H. pylori were sub-cultured in Mueller-Hilton broth supplemented with 3% sterile fetal calf serum, incubated under microaerophilic conditions at 37°C for 3 days and stored in a refrigerator after growth for subsequent use.
Primary isolation of the clinical strains was performed on Columbia blood agar (Oxoid Ltd., Basingstoke, Hants, UK) supplemented with horse blood 5% (v/v) and selectab tablet 500 mL-L (Mast Diagnostic, Merseyside, UK) which contained vancomycin 5 mg, polymycin B 25000 U, trimethoprim lactate 2.5 mg, fungizone 1.0 mg, novobiocin 2.5 mg and bacitracin 12.5 mg, final pH 7.4. H. pylori was maintained at 37°C in an automatic CO2-O2 incubator under microaerophilic conditions (85% N2, 10% CO2 and 5% O2) for 3-4 days and shaken at 150 rpm. The bacteria were subcultured and grown overnight in order to ensure logarithmic-phase growth. On the second day, cells were viewed under a phase microscope for quality control purposes. Bacterial strains were stored at -70°C in brain heart infusion broth (BHIB) (Difco, East Molesey, UK) containing 10% v/v fetal calf serum (FCS). Pylorid® (25 μg/mL) and bismuth citrate (25 μg/mL) were included as positive controls while polysorbate (fatty acid-free) Tween 80 in 50% methanol was used as negative control.
Susceptibility testing
Susceptibility was determined using the agar cup diffusion technique. A 0.1 mL aliquot of logarithmic phase broth culture of each bacterium (optical density equivalent to 107-108 cfu/mL) was used to seed sterile molten Mueller-Hinton agar (Difco, USA) medium with 5% sterile horse blood maintained at 45°C. The seeded plates were allowed to dry in the incubator at 37°C for 20 min. A standard cork borer (8 mm diameter) was used to cut uniform wells on the surface of the agar, into which was added increasing concentrations of the test extract dissolved in polysorbate (fatty acid-free) Tween 80 and 50% methanol. A pre-incubation diffusion of the extracts into the seeded medium was allowed for 1 h. Plates were incubated at 37°C in an automatic CO2-O2 incubator under micro-aerophilic conditions (85% N2, 10% CO2 and 5% O2) for 2-3 days after which diameters of zones of inhibition were measured. Since each of the extracts was reconstituted in polysorbate (fatty acid-free) Tween 80 and 50% methanol before being tested, this diluent was included in each plate as a solvent control besides the chemotherapeutic agents included as positive controls. This method is similar to previous published procedures (CitationAdeniyi, 1996; CitationAnnuk et al., 1999). The antimicrobial studies were performed in triplicate and diameters of zones of inhibition (mm) are expressed as means and standard errors as means. Student’s t-test was used to test probability at P <0.05.
Determination of minimum inhibitory concentrations
Minimum inhibitory concentrations (MICs) were performed by a modification of standard agar diffusion method procedures as previously described. Extracts were tested at various concentrations. The positive control antibiotic included was amoxicillin. The MICs were determined after 3-5 days of incubation at 37°C under microaerobic conditions. The MIC was regarded as the lowest concentration that showed the least zone of inhibition from a triplicate experiment.
Urease activity assay
The effect of the methanol extract of E. grandis on the urease activity of three H. pylori strains was investigated using the alkalimetric method (CitationHamilton-Miller & Garagan, 1979; CitationMobley et al., 1988). Fresh overnight cultures of the H. pylori strains grown in Mueller-Hinton broth supplemented with sterile fetal calf serum were centrifuged. The sediment was washed twice with 0.02 M phosphate buffer saline (PBS - pH 6.8); re-suspended again in the same PBS-pH 6.8 at OD - 560 nm) and used for urease activity assay.
In the control experiment, the bacterial suspension (0.l mL) was added to sterile test tubes containing 2.5 mL of 0.03 M PBS (pH 6.8), 0.l mL of phenol red (7 μg/mL) and 0.4 mL of urea (330 μg/L). The tubes were properly shaken and the optical density OD-560 nm and percentage transmission (% T) were recorded for a period of 1 h using a colorimeter. Approximately 10 μl of increasing concentrations of the extracts (6.5, 25, and 50 mg/mL) were added to a sterile test-tube containing similar reagents as the control experiment, and shaken. The OD-560 nm and % T were determined and recorded for a period of 1 h as in the control experiment. The OD and % T values are the urease activity values.
Cell surface hydrophobicity assay
For the determination of microbial cell surface hydrophobicity (CSH), the salt aggregation test (SAT) was performed as previously described (CitationLjungh et al., 1985). Fresh overnight cultures of H. pylori grown in Muller-Hinton broth supplemented with fetal calf serum were centrifuged for 15 min at 2000 rpm and the sediments were washed twice with 0.01 M sodium phosphate buffer (pH 7.2) containing 0.15 M NaCl. The sediments were re-suspended in the same buffer and adjusted to approximately 109 cfu of H. pylori with an absorbance of 540 nm of approximately 1.0. This was then used for SAT assay.
In the control experiment, the following molar concentrations of (NH4)2SO4 were prepared: 0.10, 0.20, 0.25, 0.30, 0.40, 1.0, 2.0, and 3.0 M, equal volumes of ammonium sulphate (0.05 mL) diluted in 0.02 M sodium phosphate buffer (pH 6.8) and bacterial suspension were mixed in wells of a flat-shaped microtiter plate. Incubation was performed at room temperature for 4 h using a modification of the standard procedure (CitationLjungh et al., 1985) to achieve visualization of possible cell aggregation. SAT was defined as positive (+) if bacterial aggregate was clearly visible, and negative if no aggregate was observed (CitationRozgonyi et al., 1990). The concentration of (NH4)2SO4 at which aggregate appeared was then recorded. The SAT titer is defined as the lowest concentration of (NH4)2SO4 at which bacteria still showed clearly visible cell aggregation. The strains were then tested for auto-aggregation in sodium phosphate buffer.
To determine the aggregation activity of H. pylori, equal volumes (0.5 mL) of bacterial suspension (109 CFU) and 25 mg/mL E. grandis methanol extract were mixed and left at room temperature for 25 min. Thereafter, 0.5 mL of this mixture was added to 0.05 mL of 0.10-3.0 M ammonium sulfate diluted with 0.02 M phosphate buffer in the wells of flat-shaped microtiter plate. After incubation for 3 h at room temperature, microbial aggregation was estimated visually as described in the control experiment.
Results and discussion
The percentage yield of the methanol extract stem bark of E. grandis after defatting with hexane was 4.26%. Phytochemical analysis of the extract indicated that it contained tannins, essential oils and saponin, but alkaloids were absent (data not shown). The antimicrobial susceptibility screening result showed that all the strains except UCH 97002 and UCH 98020 were inhibited by the extract depending on the concentration tested. In the disk diffusion assay, strain UCH 97001 exhibited the greatest susceptibility (34 mm) while strain UCH 99050 showed the least susceptibility (14 mm) (). The minimum inhibitory concentration (MIC) against the susceptible strains tested ranged between 0.39 and 1.56 μg/mL (). The antimicrobial activity of the extract compared very favorably with standard drugs ().
Table 1. Antimicrobial susceptibility of Helicobacter pylori to methanol extracts of Eucalyptus grandis. Diameter of zones of inhibition (mm) and MICs.
Urease activity of the three strains tested UCH 97001, UCH 97009 and UCH 98026 decreased with increasing concentrations of the extract (). There is, however, a slightly more pronounced urease inhibitory activity observed with UCH 97009 than the other strains. The addition of crude methanol extracts of the E. grandis extract enhanced cell aggregation of seven of the H. pylori strains studied leading to a decrease in the cell surface hydrophobicity. The SAT titer decreased from >3 to <1.5 for five of the strains, and to <3 for two of the strains ().
Table 2. Effect of methanol extracts of Eucalyptus grandis on the cell surface hydrophobility of Helicobacter pylori strains by SAT method.
Discussion
Aggressive use of antibiotics for the treatment of bacterial infections is limited by the development of resistance by the pathogenic microorganisms to many antibiotics, and thus increasing the probability of treatment failure. Helicobacter pylori infections are not an exception, as current triple therapy does not yield 100% success. In addition, the adverse events emanating from the use of anti-Helicobacter pylori drugs have led to patient non-compliance and consequent relapse of gastroduodenal disorders associated with H. pylori. Thus, the search for novel antibacterial from plant products has gathered a lot of momentum in present day research (CitationHoffman, 1997).
In Nigeria, many medicinal plants are used as antimicrobial drugs and for treatment of gastrointestinal diseases. However, records of ethnomedical use of Eucalyptus grandis are rare in the literature, but it is of interest since other Eucalyptus species extracts and oils are used for the treatment of sore throats, bacterial infections of respiratory and urinary tracts, inflammation of mucus membrane, nasal decongestion, fever bronchitis sinusitis, malaria, cough, chicken pox, and the common cold (CitationOlaniyi, 1982; CitationGill, 1992; CitationOyedeji et al., 1999).
Extracts of the stem bark of Eucalyptus grandis have strong antibacterial effects against all the susceptible strains of H. pylori tested in this investigation (with strain UCH 97001 showing the highest susceptibility). The activity of the extract may be due to the occurrence of tannins, as revealed by the phytochemical analysis. CitationAnnuk et al. (1999) isolated tannins from cowbery and bearberry, which were shown to be responsible for their high inhibitory action against H. pylori strains. Saponins, which are also present in the plant, have also been found to possess antimicrobal activities as well (CitationOsol & Hoover, 1970; Evans, 1989; CitationParker, 1989).
From the urease assay results, the addition of the extract decreased urease activity in all the strains at all concentrations tested. The urease activity decreased as the concentration of the extract increased, showing a concentration-dependent effect. The most significant decrease in urease activity was observed at the highest concentration of the extract (50 mg/mL) for all the strains. At a concentration of 25 mg/mL all the strains displayed similar urease activity of 1.5% after 10 min, and at 6.25 mg/mL, urease activity was primarily decreased in strain UCH 97009. In other words, E. grandis extracts decreased the urease activity of strain UCH 97009 more than that of UCH 97001 and UCH 98026. However, in comparison with the antimicrobial susceptibility test results, one would have expected the extract to decrease urease activity more in strain UCH 97001 since it was more susceptible to the extracts than others. Therefore, it may be concluded from the above observation that the susceptibility of H. pylori strains to E. grandis extracts is independent of its effect on urease activity. In support of this hypothesis, Nagata et al. (Citation1993) have reported that the inhibitory action of lansoprazole and its analogues against H. pylori was not related to inhibition of urease.
However, a reduction of urease activity in all H. pylori stains by E. grandis extracts is of interest from a therapeutic perspective, as urease helps the H. pylori to survive and colonize the acidic gastric mucosa by converting urea to ammonia and hence creating an alkaline milieu for the organism to survive. It further supports the concept of novel antimicrobial agents that also reduce urease activity of H. pylori strains would be worth pursuing as therapeutic agents against this pathogen.
The present work demonstrates that Eucalyptus grandis has potential as a therapeutic agent for the treatment of H. pylori. However, further investigations are needed. In vivo tests of Eucalyptus grandis extracts in rats infected with H. pylori are now underway in our laboratory.
Acknowledgements
This research work was sponsored by a Senate Research Grant from the University of Ibadan, Nigeria and the International Foundation for Science (IFS) Sweden, both to B.A.A.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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