INTRODUCTION
Cymbocarpum is represented in Iran by three species that occur naturally in the wild, including Cymbocarpum marginatum, Cymbocarpum erythraeum, and Cymbocarpum anethoides.1,2 The essential oils had been obtained from these three species of Cymbocarpum from Iran and analyzed indicating that the oils of all three plants were rich in aliphatic aldehydes.1 Furthermore, the oil of C. erythraeum, with main constituent (E)-2-decenal (52.22%), showed larvicidal activity.3 The main components of the essential oils of the fruits and herbal parts of Cymbocarpum wiedemannii were found to be aliphatic aldehydes and aliphatic acids.4
A major cause of bacterial gastrointestinal infections is Helicobacter pylori. In fact, H. pylori has been designated as a class I carcinogen by World Health Organization and its eradication has been reported to be beneficial in preventing gastric disorders specially ulcer and cancer.5 Given the extensive treatment with antibiotics for decades, the failure rates due to antimicrobial resistance range from 20% to 40% and the eradication failure rate remains as high as 5-20%.6,7,8 Regarding the previous study which indicated that methanol extract of the plant showed antibacterial activity9 and anti-H. pylori activity of some flavonoids10 the present study was designed to evaluate anti-H. pylori property of the plant extracts and its flavonoids.
MATERIALS AND METHODS
Plant material
The flowering aerial parts of C. erythraeum were collected from the East Azerbaijan province (June 2010) with voucher No. 214 at the Herbarium of Institute of Medicinal Plants, Iranian Academic Centre for Education, Culture and Research, Karaj, Iran.
General
Silica gel (70-230 mesh, F254 pre-coated plates) (Merck, Germany), reverse phase silica gel 90 (RP-18C; Fluka, Switzerland) and Sephadex LH-20 (Fluka, Switzerland) were used for isolation of compounds. Semi-preparative high-performance liquid chromatography [HPLC (RP-18C; Knauer, Germany)] and medium-pressure liquid chromatography [MPLC (Silica gel, 230-400 mesh; Butchi, Switzerland)] were used for more purification. Nuclear magnetic resonance (NMR) experiments were performed on Bruker (Billerica, USA) DRX 500 instrument [500 MHz for proton NMR (1H-NMR), 125 MHz for carbon-13 (13C-NMR)] with tetramethylsilane as an internal standard. Ultraviolet (UV) spectra were measured on Optizen (Daejeon, Korea) model 2021 UV plus. All solvents were distilled before use.
Isolation procedure
Dried aerial parts of C. erythraeum (500 g) were extracted with hexane, methanol and water-methanol (1:1) using the solvent percolation method at room temperature. Extracts were concentrated to obtain 11, 80, 34 g of hexane, methanol and methanol-water (1:1) extracts, respectively. The methanol extract was further partitioned by petroleum ether, butanol and water. The butanol fraction (8 g) was subjected to Sephadex LH20 column to afford BA-BE fractions and those suspected to contain flavonoids under UV light were loaded on another column for more purification. A sub-fractions (BA: 3980 mg, BB: 84 mg) were loaded on Sephadex LH20 column with methanol as eluent to obtain compounds 1 (8.4 mg) and 3 (12 mg). The other sub-fraction (BC: 106.6 mg) was injected to HPLC (RP-18C) eluted with different percentage of methanol-water (2:3, 1:1, and 3:2) to yield compound 2 (27 mg). In addition, the hexane extract was injected to MPLC (normal phase silica gel column) eluted with hexane-chloroform (4:1 and 0:1) to give 12 primary fractions (HA-HL). One fraction (HJ: 239 mg) was subjected to Sephadex LH20 column with chloroform-ethyl acetate-methanol (1:1:1) as eluent to provide pure compound 4 (4.1 mg).
Anti-H. pylori assay
Clinical H. pylori (HP1, HP2, and HP3) strains were used to determine antimicrobial susceptibility following previously published protocols using the disk diffusion method.11,12 Serial dilutions of the test samples were made in dimethyl sulfoxide (DMSO). Suspensions of bacteria in normal saline were prepared with the turbidity of McFarland standard No. 2 (6×108 cell/mL). Plates of non-selective blood agar were inoculated with 100 µL of each bacterial suspension and allowed to dry at room temperature. Ten µL of test samples was introduced into a sterile blank disks and deposited on the surface of the inoculated plates. Negative and positive control included blank disks impregnated with 10 µL of DMSO and amoxicillin (1 µg/mL), respectively. Plates were incubated at 37°C under microaerobic conditions and examined after 3-5 days. The mean inhibition zone diameters (IZD) ± standard deviation were recorded.
RESULTS AND DISCUSSION
The isolated compounds from aerial parts of C. erythraeum were identified as isoquercetin (1), rutin (2),13,14 along with β-sitosterol (3),15,16,17 and 2-decenol (4) based on the spectroscopic data (1H-NMR, 13C-NMR) and compared to the pertinent spectroscopic data in previously published literature (Figure 1).9
To the best of our knowledge, this is the first report on the isolation and elucidation of secondary metabolites of C. erythraeum and its anti-H. pylori activity. The susceptibility of the tested bacteria were different related to the extracts and HP3 was the most susceptible strain. The IZD of the effective test samples against H. pylori are summarized in (Table 1). The growth of all bacterial strains were suppressed by amoxicillin (1 µg/mL) with IZD of 30±0.04 mm, while DMSO showed no inhibitory activity toward the tested organisms. Methanol (464 mg/mL) and petroleum ether (226 mg/mL) extracts had inhibited one of the clinical isolates of H. pylori (HP3) with IZD of 14±0.04 and 15±0.02 mm, respectively, while compounds 1 (16 mg/mL), 2 (14 mg/mL) and the flavonoid fraction (138 mg/mL), along with other extracts were not effective against the isolated strains.
In a previous study, rutin isolated from Gardenia jasminoides exhibited antibacterial activity toward H. pylori, however this present study showed the compound not to be active against the isolated strains.18 Additionally, some flavonoids are known to be antibacterial agents.19 For example, the flavonoid-rich extract of Glycyrrhiza glabra inhibited H. pylori growth by inhibition of protein synthesis, DNA gyrase and dihydrofolate reductase10, while the flavonoid fraction of C. erythraeum was not active against examined strains, which may be presumably due to the difference in the flavonoid constituents of the plants and/or the susceptibility of bacteria. Whereas in this study, the hexane extract of the plant mainly inhibited the H. pylori strains attributed to the presence of non-polar compounds. It was reported that essential oil of C. erythraeum, which was rich in (E)-2-decenal (52.22%), was biologically active and exhibited larvicidal activity.3 In the present study, the alcoholic compound 2-decenol (4), which was isolated from hexane fraction, is likely to be one of the active constituents of this fraction as well as other constituents in this fraction. The medium-chain-length alcohol decanol, from Coriandrum sativum oil, showed antibacterial activity especially against Gram-positive strains.20,21 As a matter of fact, the relatively polar substances such as flavonoids and other polar fractions are not generally able to effectively prevent the bacterium growth, suggesting that the anti-H. pylori activity of this plant can be largely attributed to other non-polar secondary metabolites.
Conflict of Interest: No conflict of interest was declared by the authors.