¹Department of Biology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

²Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

Article Publishing History

Introduction

Helicobacter pylori (H. pylori) is an extracellular gram-negative, microaerophilic and spiral bacterium which is known as the causative agent of various types of gastrointestinal diseases and disorders such as gastric adenocarcinoma, gastric ulcer, gastritis, and lymphoma (Shrestha et al., 2012). It has been estimated that 30 to 90 percent of hospitalized patients with such gastrointestinal diseases and disorders were infected with H. pylori (Shrestha et al., 2012; Mastromarino et al., 2005; Vu and Ng, 2000). Prevalence of H. pylori in in developed and developing countries is about 40% and 90%, respectively (Suerbaum and Michetti, 2002). Gastrointestinal diseases caused by H. pylori often treated with antimicrobial agents. Prescription of ampicillin, amoxicillin, clarithromycin, metronidazole and tetracycline antibiotics is the most common way for treatment of the cases of H. pylori (Bytzer et al., 2011; Rimbara et al., 2011; Yang et al., 2014). Nevertheless, occurrence of extreme levels of antibiotic resistance in the H. pylori strains caused several concerns regarding their treatment (Bytzer et al., 2011; Rimbara et al., 2011; Yang et al., 2014; Ghotaslou et al., 2015).

Therefore, medical practitioners and also pharmacological companies have a tendency to found a suitable alternative treatment for H. pylori. Medicinal plants may be a powerful sources of antimicrobial agents which can be used for treatment of infectious diseases. The genus Thymus L. goes to the major family (Lamiaceae), and included approximately 215 species in the world. Totally, 14 species of Thymus have been described in the zone of Iran (Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab et al., 2008; Fachini-Queiro et al., 2012). T. daenensis, T. carmanicus, T. daenensis, T. persicus, T. vulgaricus and T. trautvetteri are the most commonly endemic species in Iran (Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab et al., 2008; Fachini-Queiro et al., 2012). T. daenensis subsp. daenensis is an endemic subspecies of Iran and especially high altitudes of Zagros mountains (Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab et al., 2008; Fachini-Queiro et al., 2012). Areal parts and volatile constituents of T. daenensisare used in Iranian traditional medicine.

Thymus species and especially T. daenensis are usually applied as tasty and flavoring agents in tea and also spices and medicinal drives including antimicrobial, anti-fungal, antispasmodic, and anti-inflammatory agent (Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab et al., 2008; Fachini-Queiro et al., 2012; Gautam et al., 2014). Previous investigations showed high anti-parasites, antibacterial, antiviral, antifungal, spasmolytic and antioxidant effects of Thymus species (Stahl-Biskup and Saez, 2002; Rechinger, 1982; Mojab et al., 2008; Fachini-Queiro et al., 2012; Gautam et al.,
2014).

There were no previously published data on the antimicrobial effects of Thymus daenensis on the H. pylori. Therefore, the present investigation was carried out to study the chemical composition and antimicrobial effects of the essential oil extracted from the Thymus daenensis on ten clinical isolates of H. pylori.

Material and Methods

Plant and Essential Oil Extraction

From July to August 2013, aerial parts of T. daenensis at the flowering stage were collected from the plains of the Dena mountain, Yasuj, South-west of Iran. The samples of the plant were identified and a voucher specimen was deposited at the Herbarium of Research Center of Agricultural of Shahrekord city, Iran. Essential oil of the collected T. daenensis was extracted by water distillation using the Clevenger apparatus. The essential oil was dried over anhydrous sodium sulfate and stored at 17°C until further analysis.

Table 1: Standard values for determination of sensitivity and resistance of bacteria against antibiotic agents.
Antibiotic agents	Diameter of inhibition (mm)
	Sensitive	Intermediate	Resistant
Ampicillin (10 µg)	≥17	14-17	≤13
Metronidazole (5 µg)	≥15	12-15	<12
Erythromycin (5 µg)	≥18	14-17	≤12
Clarithromycin (2 µg)	≥18	14-17	≤13
Tetracycline (30 µg)	≥19	15-18	≤14

 

Identification Of The Chemical Components

Samples were studied by the Gas Chromatography (GC, Agilent 7890 A) and Mass Spectrometry (MS, Agilent 5975 C). Identification of the chemical components was done according to the method described previously by Boruga˘et al. (2014) (Boruga˘et al., 2014). Conditions of the GC stage were as follow: temperature range of 50 to 250°C at 40°C/min with a solvent delay of 5 min. Injector of the device was kept at 250°C. Helium at a flow of 1.0 mL/min was the inert gas, and the injected volume in the splitless mode was 1 μL. The MS conditions were as follow: ionization energy of 70 eV, quadrupole temperature of 100°C, scanning velocity of 1.6 scan/s and weight range of 40-500 amu.

Bacterial Strains

Ten rough H. pylori strains were obtained from the clinical cases of gastrointestinal disorders of Alzahra Hospital, Tehran, Iran. H. pylori strains were regularly cultured on Brucella agar (Merck, Germany) supplemented with 5–7% sheep blood, amphotericin (2mg/l), polymixin-B (8mg/l), and vancomycin (6mg/l) and were incubated at 37°C for 3–7 days on the microaerophilic conditions (10% CO2 and 95% humidity). Then, H. pylori strains were approved using the morphological, Gram staining and biochemical tests (rapid urease, oxidase and nitrate) and PCR amplification of 16S rRNA gene (Forward: 5’-CTGGAGAGACTAAGCCCTCC-3’ and Reverse: 5’-ATTACTGACGCTGATTGTGC-3’) (110 bp) (Ho et al., 1991).

Table 2: Chemical components of the essential oil of the T. daenensis.
Chemical components	Retention Time (RT) (min)	Frequency (%)
beta-Phellandrene	8.26	1.92
beta-Myrcene	6.97	3.71
alpha-Thujene	5.39	2.94
alpha-Phellandrene	7.53	2.07
D-Limonene	8.04	3.56
alpha-Pinene	5.63	2.12
beta-Pinene	6.89	3.82
Terpinen-4-ol	12.55	3.45
Caryophyllene	17.32	2.71
para-Cymene	8.46	9.74
gamma-Terpinene	8.96	20.39
Thymol	16.17	42.81
Total		99.24

 

Antimicrobial Susceptibility Testing

Suspensions of the fresh cultures were made in saline and turbidity was adjusted to 1×10⁸ bacteria/ml (corresponding to turbidity with OD 0.8 at 600 nm). Two-hundred microliters of microbial suspension were placed on 50-ml Mueller Hinton agar plates containing 10% fetal calf serum (Sigma, UK) and incubated for 2–5 days at 37°C under microaerophilic conditions. H. pylori ATCC 26695 were used as quality control. Tests were done three times. Table 1 shows the standard values for susceptibility determination (CLSI, 2012). The antimicrobial effects of T. daenensis essential oil were performed by the disc diffusion method. For this purpose, sterile blank disks (6 mm) were inoculated with concentrations of 0.6, 1.25, 2.5, 5 and 10 (%v/v) essential oil and were placed on dense cultures of bacteria and incubated for 2–5 days at 37°C under microaerophilic conditions. Susceptibility of H. pylori strains were also determined against ampicillin (10 µg), metronidazole (5 µg), erythromycin (5 µg), clarithromycin (2 µg) and tetracycline (30 µg) antibiotic agents (Oxoid, UK) (CLSI, 2012).

Statistical Analysis

SPSS 20.0 software (SPSS Inc., Chicago, IL, USA) and one-way analysis of variance (ANOVA) test were used for statistical analysis. P <0.05 was considered as significant difference.

Table 3: Inhibition zone diameter for each of the ten clinical isolates of H. pylori against antibiotic agents
Antibiotic agents	Diameter of the zone of inhibition for H. pylori strains (mm)
	HP1	HP2	HP3	HP4	HP5	HP6	HP7	HP8	HP9	HP10
Metronidazole (5 µg)	0	0	0	0	0	0	16±0.4	15.4±0.2	0	0
Tetracycline (30 µg)	15.7±0.5	17.5±0.1	17±0.6	17.9±0.5	16±0.6	16.8±0.7	16.5±0.3	14.2±0.1	0	0
Ampicillin (10 µg)	12.1±0.6	0	11.3±0.8	9.1±0.7	0	19.8±0.5	16.2±0.8	0	34.8±0.8	11.7±0.6
Erythromycin (5 µg)	0	0	0	0	0	0	18.3±0.5	0	0	0
Clarithromycin (2 µg)	0	0	0	0	0	0	23.2±0.4	0	0	0

 

Results and Discussion

Table 2 represents the chemical components of the essential oil of the T. daenensis. Totally, 12 chemical components were identified in the essential oil of the T. daenensis (99.24%). Thymol (42.81%), gamma-Terpinene (20.39%) and para-Cymene (9.72%) were the most commonly identified chemical components in the essential oil of the T. daenensis. Frequency of beta-Myrcene, D-Limonene, beta-Pinene and Terpinen-4-ol were 3.71%, 3.56%, 3.82% and 3.45%, respectively.

Table 3 represents the inhibition zone diameter for each of the ten clinical isolates of H. pylori against antibiotic agents. Results showed that H. pylori strains had the highest levels of sensitivity against tetracycline and ampicillin antibiotics. Most resistance was seen for the erythromycin, clarithromycin and metronidazole antibiotics. Statistically significant differences (P <0.05) were seen between the zone of inhibition and types of antibiotics.

Table 4: Inhibition zone diameter of the various concentrations of the T. daenensis essential oil against H. pylori.
Essential oil concentrations	Diameter of the zone of inhibition for H. pylori strains (mm)
	HP1	HP2	HP3	HP4	HP5	HP6	HP7	HP8	HP9	HP10
0.6%	0	5.8±0.4	6±0.5	0	5.3±0.8	0	0	4.1±0.2	11.2±0.6	5.5±0.2
1.25%	0	5.3±0.9	7.8±0.1	7±0.0	6.7±0.3	0	0	5.2±0.8	14.3±0.4	5.2±0.5
2.5%	4.6±0.3	8.3±0.0	9.7±0.4	10.2±0.5	9.1±0.1	11.4±0.5	8.8±0.2	7.5±0.5	15.5±0.1	8.4±0.3
5%	5.3±0.5	13.1±0.5	13.2±0.1	12.8±0.6	11.6±0.2	12±0.9	14.1±0.1	10.2±0.4	16.7±0.2	11.6±0.4
10%	6.2±0.7	15.7±0.2	13.6±0.2	14.3±0.4	14±0.5	17.1±0.2	16±0.3	12.3±0.4	18.6±0.5	13.2±0.8

 

Table 4 represents the inhibition zone diameter of the various concentrations of the T. daenensis essential oil against H. pylori strains. We found that increase in the concentration of essential oil cause its higher antimicrobial effects on the H. pylori strains isolated from the clinical cases of gastrointestinal disorders (P <0.05). Inhibition zone diameter increased from 0 to 18.6±0.5 mm which showed that the anti-H. pylori effects of T. daenensis essential oil is dose depended.

Failure of majority of therapeutic options and especially ampicillin, amoxicillin, clarithromycin, metronidazole and tetracycline antibiotics caused us to study the antimicrobial effects of H. pylori strains against T. daenensis essential oil. The results of the present investigation showed that the T. daenensis essential oil had a high antimicrobial effects on the H. pylori strains isolated from clinical cases of gastrointestinal disorders. Several studies have been approved the anti-H. pylori effects of some medicinal plants. Falsafi et al. (2014) (Falsafi et al., 2014) reported that Satureja bachtiarica essential oil showed strong antibacterial activity against clinical isolates of H. pylori (17.6 ± 1.1 mm and 0.035 ± 0.13 μl/ml). They showed that anti-H. pylori effects of S. bachtiarica is due to its high carvacrol content. Esmaeili et al. (2012) (Esmaeili et al., 2012) reported that Shoya powder and essential oils of Thymus vulgaris and Eucalyptus globulus had significant effects on the H. pylori. They showed that Minimum Inhibitory Concentration (MIC) of the T. vulgaris and E. globulus were 10.8 and 46.4 (µg/ml) respectively.

The antimicrobial effects of the T. daenensis essential oil is due to the presence of some active constituents with their hydrophobicity which enables them for rupturing cell membranes and intrastructures. We found that Thymol (42.81%), gamma-Terpinene (20.39%) and para-Cymene (9.72%) were the most commonly identified chemical components in the essential oil of the T. daenensis. Previous study in Romania (Grigore et al., 2010) showed that the most commonly detected components in the essential oil of the Thymus were p-cymene, ã-terpinene and thymol which was similar to our findings, while those of Morocco (Imelouane et al., 2009) and Spain (Ballester-Costa et al., 2013) are entirely different with us. P-cymene, ã-terpinene and thymol were the most commonly detected components in previous studies conducted on Iran (Pirbalouti et al., 2013), Poland (Kowalski and Wawrzykowski, 2009), Italy (De Lisi et al., 2011) and Spain (Rota et al., 2008). Probably types of samples, method of sampling, genus of plants, and differences in the climate and weather of various geographical regions are the main factors which may affected on the chemical components of Thymus plants of different studies.

Antimicrobial effects of T. daenensis essential oil is depends on its chemical compositions. It seems that high content of the phenolic compounds (thymol) and terpene hydrocarbons (ã-terpinene) is the main factor for the high anti-H. pylori effects of T. daenensis. Previous investigations showed that para-cymene does not display any antimicrobial activities alone (Dorman and Deans, 2000), while it has a strong antibacterial effects in relation with thymol and ã-terpinene (Delgado et al., 2004; Gallucci et al., 2009).

Conclusion

In conclusion, we found that T. daenensis essential oil has a high antimicrobial effects on the H. pylori strains of human clinical infections. As it showed, Thymol, gamma-Terpinene and para-Cymene were the most commonly identified chemical components in the essential oil of the T. daenensis. In compare with the findings of other researchers, Preparation of good conditions for growth of T. daenensis full from these chemical components will help us to present an efficient antibiotic agent for treatment of the cases of H. pylori-infection.

Acknowledgements

Authors would like to thanks from Dr. Tahereh Falsafi, Dr. Mohaddase Mahboubi, Dr. Behzad Hamedi, Prof. Ebrahim Rahimi and Prof. Hassan Momtaz for their important clinically support. This work was supported by the Alzahra University, Tehran, Iran.

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