Abstract
The chemical composition of the essential oil obtained by hydrodistillation of Pistacia vera L. gum was analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). Twenty compounds were identified, representing 99.5% of the total components. α-Pinene, β-pinene, and α-thujene were found to be the major constituents. The bacteriostatic activity of the essential oil against 12 clinical isolates of Helicobacter pylori was determined using the hole-plate method. All isolates were sensitive to the essential oil, and the Minimum Inhibition Concentration (MIC) was 1.55 mg/ml for all isolates using the agar dilution method.
Introduction
Three species of Pistacia grow in Iran, of which Pistacia vera L. is widely cultivated in different parts of Iran (CitationMozaffarian, 1996). The resin of the plant is collected and sold in local markets as a substitute for mastic gum, an exudate of P. lentiscus, which does not grow in Iran. The resin is used in traditional medicine as expectorant, sudorific, carminative, and antitussive (Zargari, Citation1997). Recently, the antimicrobial activity of the essential oil obtained from resin of P. lentiscus has been reported against several Gram-positive and Gram-negative bacteria and three fungi (Mgiatis et al., Citation1999). In clinical trials, mastic gum has been effective in the treatment of benign gastric ulcers (Huwez & Al-Habbal, Citation1986) and duodenal ulcers (Al-Habbal et al., Citation1986). Furthermore, cytoprotective and mild antisecretory activities were reported for the mastic tested in rats (Al-Said et al., Citation1986). Although the anti-Helicobacter activity of the gum (Huwez et al., Citation1998s) together with analysis of the gum essential oil of P. lentiscus (Boelens & Jimenez, Citation1991; Papageorgiou et al., Citation1991; Mgiatis et al., Citation1999) and its antimicrobial activity (Mgiatis et al., Citation1999s) have been reported, no chemical investigation of the essential oil of P. vera and the anti-Helicobacter activity of the essential oil of any Pistacia species have been reported so far.
In the current study, the chemical composition of the gum oil of P. vera was determined by GC-MS, and its inhibitory activity against fresh clinical isolates of Helicobacter pylori are reported.
Materials and methods
Plant material and essential oil isolation
The gum of P. vera was collected near Damghan (a city in Semnan province, Iran) in April 2001. The plant was identified by the Department of Botany, Ferdowsi University (Mashhad, Iran). A voucher specimen (no. 554) was deposited in the Herbarium of the Faculty of Pharmacy, Mashhad University of Medical Sciences.
The oil was obtained by hydrodistillation using a Clevenger-type apparatus for 3 h to give a colorless oil. It was dried over anhydrous sodium sulfate and stored at 4–8°C in the dark.
Identification of oil components
GC-MS analysis was carried out on a Varian 3400 GC-MS system equipped with a DB-5 fused silica column (30 m × 0.25 mm i.d., film thickness 0.25 µm). The oven temperature was held at 60°C for 5 min and programmed to 275°C at a rate of 4°C/min; injector and transfer line temperatures were 280°C; the carrier gas was helium with a linear velocity of 31.5 cm/s; split ratio, 1:10; ionization energy, 70 eV; scan time, 1 s; mass range, 35–400 amu.
Anti-Helicobacter assay
Twelve randomly selected clinical isolates from antral biopsies from children and adults were used in this study. Clinical isolates of H. pylori were obtained from the Ghaem Hospital (Mashhad, Iran). Primary isolation was performed on selective Colombian agar medium (100 ml) containing fetal calf serum (5% v/v), L-cysteine (0.1% w/v), sheep blood (5% v/v), trimetoprim, vancomycine and amphotricine (0.01% w/v for each), and β-cyclodextrine (0.1% w/v). The inhibitory activity of the oil was evaluated using the hole-plate method (Ieven et al., Citation1979s). Holes were made on plates containing Colombian agar and serial dilutions of the essential oil dissolved in methanol poured in holes (three holes for each dilution). The plates were inoculated with 0.1 ml of H. pylori suspension in brain heart infusion broth and incubated for 5–7 days under micro-aerobic conditions at 37°C. Metronidazole and amoxicilline, in concentrations of 5 and 25 µg/disk, were used as positive controls, and methanol was used as negative control. The zones of inhibition were measured and expressed in millimeters. The MIC of the essential oil was determined using an agar dilution method (Shahamat et al., Citation1991s). Various concentrations of the essential oil mixed with Tween 80 (3:2) were poured in the molten (45°C) Colombian agar medium followed by inoculation of the plate with 0.1 ml of H. pylori suspension in brain heart infusion broth. The plates were incubated at 37°C under micro-aerobic atmosphere for 7 days. The MIC was expressed as the lowest concentration of the essential oil that inhibited visible growth.
Results and Discussion
The yield (v/w) of the essential oil was 17%. GC-MS analysis indicated 20 compounds (), representing 99.3% of the total components. The retention indices for all components were determined according to the Van den Dool's method using n-alkanes as standard (Van Den Dool & Kratz, Citation1963s). Compounds were identified by comparison of retention indices (RII, DB-5) with those reported in the literature (Adams, Citation1995s) and by comparison of their mass spectra with the Wiley library (Massada, Citation1976) or with published mass spectra (Adams, Citation1995s). The major constituents of the essential oil of the gum were α-pinene (60.7%), β-pinene (14.7%), and α-thujene (7.3%). The other notable minor components were limonene (2.5%), camphene (1.8%), and cis-verbenol (1.7%). High content of α-pinene was also found in the oil gum of P. lentiscus (Mgiatis et al., Citation1999). Considering that the gum of P. lentiscus exhibited anti-Helicobacter activity (Huwez et al., Citation1998), the oil showed antimicrobial activity (Mgiatis et al., Citation1999), and α-pinene was the major constituent of the oil from P. lentiscus and P. vera prompted us to investigate the inhibitory activity of the gum oil of P. vera against H. pylori. The oil demonstrated a high inhibitory activity against H. pylori in a dose-dependent manner. Serial dilutions of the oil in methanol were tested including 1/10, 1/20, 1/40, 1/80, and 1/100. Results indicated zone of inhibitions ranging from 7 to 24 mm in all 12 clinical isolates. All isolates were sensitive to metronidazole and amoxicillin as positive controls (). The MICs for all isolates was 1.6 µl/ml. The inhibitory activity of the oil could be attributed to the high content of α-pinene. It has been reported that α-pinene possessed antimicrobial activity (Knobloch et al., Citation1989). Furthermore, the gum of P. lentiscus was highly effective to eradicate the H. pylori–infected patients (Huwez et al., Citation1998), and α-pinene was the major constituent of gum oil of P. lentiscus (Mgiatis et al., Citation1999). All these data support the idea that the inhibitory activity of the oil could be due to α-pinene. Further studies are underway to test the antimicrobial activity of α-pinene, β-pinene, and α-thujene against H. pylori.
Table 1 Composition of the essential oil of Pistacia vera L. gum.
Table 2 Bacteriostatic activity (zones of inhibition in mm) of the gum oil of Pistacia vera against Helicobacter pylori.
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