Chemical composition and anti-Helicobacter pylori effect of Satureja bachtiarica Bunge essential oil.
Resistance of H. pylori strains to common antibiotics has been developed in different parts of the world and continues to increase. It is important to investigate the novel and efficient anti-H. pylori drugs, among which the plants would be suitable sources.
Satureja bachtiarica Bunge is traditionally used as antimicrobial agent. In this study, we evaluated the antibacterial activity of S. bachtiarica Bunge essential oil against 10 clinical isolates of Helicobacter pylori by disc diffusion and agar dilution methods. The chemical composition of essential oil was analyzed by GC and GC-MS. Carvacrol (45.5%) and thymol (27.9%) were the primary constituents of oil, followed by p-cymene (4.4%), and [gamma]-terpinene (4.0%). S. bachtiarica essential oil showed strong antibacterial activity against clinical isolates of H. pylori (17.6 [+ or -] 1.1 mm and 0.035 [+ or -] 0.13 [micro]l/ml). Carvacrol, as the first main component, had a significant role in this effect, whereas in the presence of thymol, the antibacterial effect of carvacrol was reduced. Therefore, S. bachtiarica essential oil can be applied as an alternative agent for treatment of H. pylori infections. More studies would be required to better clarify its mechanism of action on H. pylori.
Satureja bachtiarica Bunge
Helicobacter pylori is the most common cause of gastric and duodenal ulcers. It is also associated with adenocarcinoma and Mucosa Associated Lymphoid Tissue lymphoma (de Bernard and Josenhans 2014). Successful treatment of H. pylori infections consists on triple therapy, comprising a proton pump inhibitor and any of the two antibiotics such as amoxicillin (AMX), clarithromycin (CLA), metronidazole (MNZ) and tetracycline (TET). Nevertheless, H. pylori resistance to most of these commonly used antibiotics is raising worldwide (lwanczak and Iwanczak 2012). There is also the resistance problem related to inappropriate and extensive use of such drugs (Megraud 2012).
Plants may be the alternative main source of raw drugs and folk medicines since they are known to contain active metabolites, which are useful in treating various infectious diseases with no or less toxicity (Cowan 1999).
In Iran, a large number of herbal extracts are used in folk medicine to treat various types of digestive disorders, and several studies have documented the beneficial effects of Iranian plants in the prevention of gastric injury. Among them, several natural medicinal plants, herbs, have been proved to have antimicrobial activity against H. pylori (Biglar et al. 2012; Nariman et al. 2004).
The genus Satureja consists of 15 species in Iran and nine out of them are endemic. They include: Satureja edmondi, S. kallarica, S. sanhandica, S. khuzestanica, S. isophylla, S. intermedia, S. atropatana, S. rechingeri and S. bachtiarica (Mozaffarian 1997). Satureja bachtiarica Bunge with local name "Merzeh Koohi" is an aromatic medicinal plant (Lamiaceae family) which is traditionally used as an analgesic and antiseptic agent (Gulluce et al. 2003; Zargari 1992). Different parts of S. bachtiarica including flowers, leaves and stems are used as herbal tea to treat various ailments, such as cramps, muscle pains, nausea, indigestion, diarrhea and infectious diseases (Gulluce et al. 2003). This genus originates from the Mediterranean region (Oke et al. 2009) and is distributed in the Southern and southwest regions of Iran. There are some reports that evaluate the biological activity of S. bachtiarica including its wide pharmacological effects, as well as immune enhancing effect (Ghasemi Pirbalouti et al. 2011b). S. bachtiarica may also improve the amyloidal [beta] induced memory impairment in Alzheimer disease (Ghasemi Pirbalouti et al. 2011 b; Soodi et al. 2012) and may have antioxidant activity (Hashemi et al. 2011). The antimicrobial activities of S. bachtiarica were confirmed against Pseudomonas aeruginosa (Ghasemi Pirbalouti and Dadfar 2013; Sefidkon et al. 2005; Teimori 2009), Bacillus subtilis, Micrococcus luteus, Klebsiella pneumonia, Klebsiella oxytoca and Staphylococcus areus (Teimori 2009), Candida albicans (Ghasemi Pirbalouti et al. 2009; Rohi Boroujeni et al. 2012), Streptococcus iniae (Ghasemi Pirbalouti et al. 2010), Rhizoctonia solani (Foroughi et al. 2013), Escherichia coli (Ghasemi Pirbalouti et al. 2010; Sureshjani et al. 2013), Listeria monocytogenes (Ghasemi Pirbalouti et al. 2010), Vibrio parahaemolyticus, Vibrio harveyi (Ghasemi Pirbalouti et al. 2011a).
S. bachtiarica is containing an oil which has unique composition; therefore, in recent years, it has been the subject of many researches (Ghasemi Pirbalouti and Dadfar 2013; Sefidkon et al. 2005; Teimori 2009).
Essential oil of S. bachtiarica is composed of phenolic compounds such as thymol, [gamma]-terpinene and carvacrol (Ghasemi Pirbalouti and Dadfar 2013; Sefidkon et al. 2005; Teimori 2009). However, ecological conditions such as altitude, temperature, soil, moisture, growth stage can affect the chemical composition of S. bachtiarica oil (Ahmadi et al. 2009), thereof, it is indispensable to measure the chemical composition of oil and assess its biological action.
To our knowledge, there are no published reports about the antibacterial activity of S. bachtiarica essential oil against Helicobacter pylori.
So, in this study, we evaluated the chemical composition of S. bachtiarica essential oil and its antibacterial activity against clinical isolates of Helicobacter pylori.
Materials and methods
Plant materials and extraction of S. bachtiarica essential oil
Aerial parts of Satureja bachtiarica at the beginning of the flowering stage, were collected from Chaharmahal Va Bakhtiari province, Southwest of Iran in July 2013. The samples of the plant were identified and a voucher specimen was deposited at the Herbarium of Research Center of Agricultural of Chaharmahal VA Bakhtiari province, Iran (No. 1999). The oil was extracted by water distillation in Clevenger type apparatus. The oil was dried over anhydrous sodium sulfate and stored at 17[degrees]C until further analysis.
Oil chromatographic analysis
Essential oil was analyzed by GC-FID (Agilent 7890 A) and GCMS (Agilent 5975 C) with a column of HP-5 MS (30 m x 0.25 mm i.d., film thickness, 0.25 [micro]m) and detector. The sample was injected by splitting and the split ratio was 1:100. The oven temperature programmed as follows: 60-250[degrees]C at 5[degrees]C/min, then held for 10 min at 250[degrees]C. Helium was used as the carrier gas at a flow rate of 1.1 ml/min. The injector and detector temperatures were 250[degrees]C and 260[degrees]C, respectively. Identification of components was achieved from their GC retention indices (RI) relative to n-alkanes and by computer search using libraries of Wiley275.L and Wiley7n.1, as well as comparisons of the fragmentation pattern of the mass spectra with data published in the literature. Quantization of major constituents of the oils were compared by the retention time and RIs of related authentic components (thymol, carvacrol, p-cymene, [gamma]-terpinene) (Adams 2001; National Institute of Standard and Technology 1998).
Chemical materials and antibiotics
Thymol, carvacrol were purchased from Merck (Darmstadt, Germany) while the antibiotic discs were purchased from Rosco Diagnostica A/S, Taastrupgaardsvej 30 DK-2630 Taastrup). The antibiotic discs were metronidazole (4 [micro]g), ampicillin (10 [micro]g), tetracycline (30 [micro]g), erythromycin (15 [micro]g) and clarithromycin (15 [micro]g).
Helicobacter pylori strain
Ten H. pylori strains were selected from a collection of clinical isolates from Alzahra University, Tehran, Iran. This selection was made on the basis of their resistance pattern to various antibiotics, previously evaluated. The H. pylori strains were routinely cultured on Brucella agar (Biolife; Albimi, Italiana S. r. L. Viale Monza 272-20178-Milano-Italia) supplemented with 5-7% sheep blood, polymixin-B (8 mg/l), amphotericin (2 mg/l) and vancomycin (6 mg/l) and were incubated for 3-7 days under microaerophilic conditions (10% C[O.sub.2] and 95% humidity) at 37[degrees]C. The H. pylori strains were confirmed by morphological colonies, Gram staining, biochemical test (oxidase, rapid urease, and nitrate) and PCR reaction using the primers for H. pylori specific 16sRNA and ureC as previously described (Falsafi et al. 2007).
Antibacterial activity evaluation by disk diffusion method
Suspensions of the fresh cultures were made in saline and turbidity was adjusted to 1 x [10.sup.8] bacteria/ml (corresponding to turbidity with OD 0.8 at 600 nm). In each case, 200 [micro]l of the appropriate microbial suspension was placed on 50-ml Mueller Hinton agar plates containing 10% fetal calf serum (Gibco/BRL, Paisley, UK) and spread evenly in all directions then incubated at 37[degrees]C under microaerophilic conditions for 2-5 days. H. pylori ATCC 26695 were used as quality control. Each test was repeated as triplicate. The cutoff values for susceptibility determination corresponded to universal standards (CLSI 2009).
The antimicrobial activity of S. bachtiarica essential oil and its components were performed by the disc diffusion method. For this purpose, sterile blank disks (6 mm) were inoculated with plant oil containing 0.6,1.25,2.5,5 and 10 (% v/v) essential oil, thymol or carvacrol and were placed on the surface of the bacterial lawn and incubated at 37[degrees]C under microaerophilic conditions for 2-5 days.
Antibacterial activity evaluation by agar dilution method
The agar dilution method was used according to instructions approved by the CLSI (CLSI 2006). For this purpose, the oil was added in 2-fold decreasing concentrations to Mueller Hinton agar containing 10% serum. Fresh bacterial suspensions (equivalent to 1 x [10.sup.8] bacteria/ml) were used to inoculate the plates and incubated as described above. To enhance the essential oil solubility, the essential oil was dissolved in 0.5% (v/v) tween-20% and added into the agar. Inoculated plates were incubated as described above and minimum inhibitory concentrations were recorded. Controls of bacteria without the oil or oil without bacteria were also included in this study.
The SPSS for Windows (1MB, SPSS 22, Inc, Chicago, IL) software was used for data and statistical analysis. Numerical data were analyzed using the one-way analysis of variance (ANOVA) in order to compare the mean values. A probability equal to or less than 5% was considered statistically significant.
Results and discussion
The chemical analysis of S. bachtiarica oil showed the presence of 15 components that present 91% of total oil composition. Carvacrol (45.5%), thymol (27.9%) were the main components of oil, followed by p-cymene (4.4%), [gamma]-terpinene (4.0%), [alpha]-pinene (1.5%), 1,8-cineole (1.3%), [alpha]-terpinene (1.2%) and E-caryophyllene (1.1%), respectively (Table 1). Other components such as [alpha]-thujone (0.9%), camphene (0.8%), [beta]-pinene (0.4%), myrcene (0.9%), terpinolene (0.4%), terpinene-4-ol (0.8%) and geranyl acetate (0.3%) were traced in S. bachtiarica oil (Table 1).
As mentioned before, the chemical composition of S. bachtiarica oil is affected by ecological conditions (Ahmadi et al. 2009). Carvacrol (62.3%), p-cymene (21.2%), [gamma]-terpinene (5.2%) were the main components of S. bachtiarica oil from Khoram Abad province, while carvacrol (25.8%), p-cymene (23.2%), menthone (18.5%), thymol (1.3%) were found in S. bachtiarica oil from Shahr-E-Kord (Ahmadi et al. 2009) and carvacrol (26.4%), thymol (20.6%) and linalool (14.2%) were isolated from S. bachtiarica oil from Ardebil province (Teimori 2009). Furthermore, the chemical composition of the oils is affected by harvesting time (Mahboubi and Haghi 2008). It has been reported; carvacrol (25.8%) and p-cymene (25.2%), p-menth-3-en-8-ol (18.5%), borneol (6%) and thymol (5%) as the main components of S. bachtiarica oil at the full flowering stage while p-cymene (36.5%), carvacrol (20%), thymol (19.2%) and [gamma]-terpinene (9.1%) were found in the essential oil from before flowering stage of plant (Sefidkon et al. 2005).
The drying method of plant can affect on chemical composition of S. bachtiarica oil. Carvacrol (31.2-42.2%), [gamma]-terpinene (10.9-18.3%), thymol (11.7-19.4%) and p-cymene (8.2-14.1%) were the main components of oils from the plant which was dried by sun-drying, shade-drying, oven-drying at 45[degrees]C, 65[degrees]C and freeze-drying. The oven drying at 45[degrees]C is the most suitable method for drying the S. bachtiarica (Ultee et al. 2002).
Therefore, determining the chemical composition of S. bachtiarica oil before the screening of its antibacterial activity is critical. In this study, carvacrol was the first main component of S. bachtiarica oil followed by thymol, p-cymene and [gamma]-terpinene; respectively. The result of the chemical composition of our study was opposed to Ghasemi Pirbalouti and Dadfar (2013) study, where p-cymene (40.5%), thymol (17.9%), carvacrol (7.8%) and [gamma]-terpinene (6.9%) were the main components of S. bachtiarica oil (Ghasemi Pirbalouti and Dadfar 2013).
The susceptibilities of 10 H. pylori clinical isolates to selected antibiotics are demonstrated in Table 2. Most resistance was observed against Macrolide (erythromycin and clarithromycin) followed by metronidazole and [beta]-lactam (Ampicilin).
The antibacterial activity of S. bachtiarica oil against clinical isolates of H. pylori (Table 2) by disc diffusion method showed the inhibition zone diameter (mm). This was increased from 8.6 [+ or -] 0.93 to 21 [+ or -] 1.3 in the presence of 0.6-10% of S. bachtiarica oil. Therefore, the antibacterial activity was increased dose dependently. The antibacterial activity of thymol and carvacrol against clinical isolates of H. pylori showed that the antibacterial activity of carvacrol was higher than thymol against H. pylori. The antibacterial activity of thymol was lower (8.3 [+ or -] 1.3 mm) than S. bachtiarica oil (17.6 [+ or -] 1.1 mm) while this effect was higher for carvacrol (29.1 [+ or -] 3.4 mm) than S. bachtiarica oil. The antibacterial activity about the combination of thymol and carvacrol (23.3 [+ or -] 3.6 mm) was higher than S. bachtiarica oil (17.6 [+ or -] 1.1 mm) and thymol alone. The antibacterial activity of carvacrol (29.1 [+ or -] 3.4 mm) was higher than thymol and carvacrol combination and the mean difference was significant at 0.05 level (p < 0.05). The inhibition zone (IZ) diameter of S. bachtiarica oil (17.6 [+ or -] 1.1 mm) and thymol and carvacrol combination was not significant (p > 0.05). The IZ for antibiotics was lower than the S. bachtiarica oil and carvacrol.
The results of the disc diffusion method were confirmed by agar dilution assay (Tables 3 and 4). The meaning of MIC values of thymol and carvacrol combination (0.0175 [+ or -] 0.006 [micro]l/ml) and carvacrol (0.0181 [+ or -] 0.029 [micro]l/ml) was lower than the S. bachtiarica oil (0.035 [+ or -] 0.13 [micro]l/ml) and thymol (0.043 [+ or -] 0.024 [micro]l/ml). Although, the results of the disc diffusion method and agar dilution assay are comparable, but there are three subsets of compounds, including 1-carvacrol, thymol plus carvacrol, 2- S. bachtiarica oil and 3- thymol in disc diffusion method and two subsets (1- thymol plus carvacrol, carvacrol; 2- S. bachtiarica oil and thymol) of compounds in agar dilution.
The results of antimicrobial screening showed that the antibacterial activity of S. bachtiarica oil was higher than current antibiotics, including metronidazole, clarithromycin and ampicillin. Therefore, S. bachtiarica oil may be a suitable alternative for treatment of H. pylori infections.
The antibacterial activity of S. bachtiarica essential oil relates to phenolic compounds, especially thymol and carvacrol. The anti-Helicobacter pylori effect of carvacrol was higher than essential oil. Carvacrol has a hydroxyl group with potency as trans membrane carrier for monovalent cations (Ultee et al. 2002), also it destroys the proton motive force, enzymes and many other essential macromolecules (Burt 2004).
Although, thymol is reported as a strong antimicrobial agent (Veldhuizen et al. 2006) and the hydroxyl group and delocalized electrons of thymol are responsible for damaging the cytoplasmic membrane (Sikkema et al. 1995), the citrate metabolic pathway (Di Pasqua et al. 2010), interacting with membrane and intracellular proteins and changing the membrane permeability, the leakage of potassium ions, ATP and carboxy fluorescein (Xu et al. 2008) of other bacteria. But thymol showed little activity against clinical isolates of H. pylori also, the presence of thymol in S. bachtiarica essential oil decreases the activity of thymol. Therefore, it can be concluded that thymol has significant anti-Helicobacter activity.
There are confusing reports about the synergistic or antagonistic effects of thymol and carvacrol. Thymol and carvacrol had been reported to have a synergistic effect (Didry et al. 1994) but another report showed the toxicity of carvacrol was reduced in the presence of thymol, suggesting an antagonistic effect (Chiasson et al. 2001). Our results confirm the antagonistic activity of thymol and carvacrol against Helicobacter pylori. Other components of S. bachtiarica essential oil are responsible for its antimicrobial activity. The antimicrobial activity of caryophyllene (Kim et al. 2008; Lu et al. 2006) has been confirmed while [gamma]-terpinene (Cox et al. 2001; Juven et al. 1994), 1,8-cineole, [alpha]-pinene, [beta]-pinene (Andrews et al. 1980), p-cymene (Andrews et al. 1980; Ultee et al. 2002) showed no antibacterial activity against Gram negative bacteria. Furthermore, it is reported that some components with no antibacterial activity in the presence of some antimicrobial agent showed a synergistic effect, for example, the presence of p-cymene along with carvacrol may enhance its antimicrobial activity of oil (Ultee et al. 2002). Therefore, the major and minor components of oil or synergistic or antagonistic effects of these components are responsible of antibacterial activity of S. bachtiarica essential oil against clinical isolates of H. pylori. At the first of further study, it is essential to standardize of S. bachtiarica essential oil with higher amount of carvacrol. Other pharmacological, toxicological, clinical and formulation studies have an important role in demonstrating the efficacy of S. bachtiarica essential oil.
Conflict of Interest
There is no conflict of interest.
Received 4 April 2014
Revised 6 September 2014
Accepted 20 November 2014
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Tahereh Falsafi (a) Parisa Moradi (a), Mohaddese Mahboubi (b), *, Ebrahim Rahimi (c), Hassan Momtaz (d), Behzhad Hamedi (e)
(a) Department of Biology, Alzahra University, Tehran, Iran
(b) Department of Microbiology, Medicinal Plant, Research Center of Barij, Kashan, Iran
(c) Department of Food Hygiene and Public Health, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
(d) Department of Microbiology, Faculty of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
(e) Department of Medicinal Plants, Faculty of Agriculture, Islamic Azad University-Shahrekord Branch, Shahrekord, Iran
* Corresponding author. Tel.: +98 8644465112; fax: +98 8644465187. E-mail address: firstname.lastname@example.org, email@example.com (M. Mahboubi).
Table 1 Chemical composition of Satureja bachtiarica essential oil. Compound RI * RI ** RT *** (%) **** [alpha]-Thujone 930 0.9 [alpha]-Pinene 936 1.5 Camphene 951 0.8 [beta]-Pinene 984 0.4 Myrcene 994 0.9 [alpha]-Terpinene 1017 1.2 p-Cymene 1025 1020 13.98 4.4 1,8-Cineole 1031 1.3 [gamma]-Terpinene 1058 1054 15.2 4.0 Terpinolene 1099 0.4 Terpinene-4-ol 1173 0.8 Thymol 1292 1296 22.99 27.9 Carvacrol 1305 1298 23.21 45.5 Geranylacetate 1380 0.3 E-caryophyllene 1413 1.1 * RI, retention index. ** RI for authentic samples. *** RT, retention time. **** (%), relative percentage obtained from peak area. Table 2 Disk diffusion results of five antibiotics against 10 clinical isolates of H. pylori. Inhibitory zone (mm) for each of 10 strain Antibiotic HP1 HP2 HP3 HP4 HP5 Metronidazole 0 0 15 16 0 Tetracycline 0 0 14 16 16 Ampicillin 11 34 0 16 19 Erythromycin 0 0 0 18 0 Clarithromycin 0 0 0 23 0 Inhibitory zone (mm) for each of 10 strain Antibiotic HP6 HP7 HP8 HP9 HP10 Metronidazole 0 0 0 0 0 Tetracycline 16 17 17 17 15 Ampicillin 0 9 11 0 12 Erythromycin 0 0 0 0 0 Clarithromycin 0 0 0 0 0 Cut off Antibiotic R I s Metronidazole <12 12-15 [greater than or equal to] 15 Tetracycline [less than or 14-17 [greater than equal to] 12 or equal to] 18 Ampicillin [less than or 15-18 [greater than equal to] 14 or equal to] 19 Erythromycin [less than or 14-17 [greater than equal to] 13 or equal to] 17 Clarithromycin [less than or 14-17 [greater than equal to] 13 or equal to] 18 Concentration of antibiotics is included: metronidazole (4 [micro]g); tetracycline (30 [micro]g): ampicillin (10 [micro]g): erythromycin (15 [micro]g): clarithromycin (15 [micro]g): I: intermediate; R: resistance; S: sensitive. Table 3 The inhibition zone (IZ) diameter (mm) of different components against clinical isolates of H. pylori. Concentration Component 0.6% 1.25% S. bachtiarica oil 8.6 [+ or -] 0.93 12 [+ or -] 0.94 Thymol NE NE Carvacrol 3.9 [+ or -] 0.83 10.4 [+ or -] 1.14 Thymol + carvacrol *** NE 5 [+ or -] 1.07 Metronidazole -- -- Tetracycline -- -- Ampicillin -- -- Erythromycin -- -- Clarithromycin -- -- Concentration Component 2.5% 5% S. bachtiarica oil 16.5 [+ or -] 0.97 21 [+ or -] 1.3 Thymol 5.1 [+ or -] 1.1 12.4 [+ or -] 0.7 Carvacrol 20.5 [+ or -] 1.1 40.7 [+ or -] 1.2 Thymol + carvacrol *** 14.1 [+ or -] 0.86 32.6 [+ or -] 1.4 Metronidazole -- -- Tetracycline -- -- Ampicillin -- -- Erythromycin -- -- Clarithromycin -- -- Concentration Component 10% Ab * S. bachtiarica oil 29.9 [+ or -] 0.9 -- Thymol 24.2 [+ or -] 1 -- Carvacrol 69.9 [+ or -] 0.34 -- Thymol + carvacrol *** 67.6 [+ or -] 0.94 -- Metronidazole -- 3.1 [+ or -] 2.1 Tetracycline -- 12.8 [+ or -] 2.1 Ampicillin -- 10.2 [+ or -] 3.1 Erythromycin -- 1.8 [+ or -] 1.8 Clarithromycin -- 2.3 [+ or -] 2.3 Concentration Component Total Subsets ** S. bachtiarica oil 17.6 [+ or -] 1.1 17.6 (bc) Thymol 8.3 [+ or -] 1.3 8.3 (c,d) Carvacrol 29.1 [+ or -] 3.4 29.l (a) Thymol + carvacrol *** 23.3 [+ or -] 3.6 23.3 (c,b) Metronidazole 3.1 [+ or -] 2.1 3.1 (d) Tetracycline 12.8 [+ or -] 2.1 12.8 (b,c,d) Ampicillin 10.2 [+ or -] 3.1 10.2 (c,d) Erythromycin 1.8 [+ or -] 1.8 1.8 (d) Clarithromycin 2.3 [+ or -] 2.3 2.3 (d) Ab * : concentration of antibiotics are including: metronidazole (4 [micro]g); tetracycline (30 [micro]g); ampicillin (10 [micro]g); erythromycin (15 [micro]g); clarithromycin (15 [micro]g); NE: no effect; (-): not determined. *** Equal to the combination of thymol and carvacrol in S. bachtiarica oil. ** The different letters (a, b. c, d) indicate significant at the level [less than or equal to] 0.05 by Tukey's Range test. Table 4 The MIC values ([micro]l/ml) of Satureja bachtiarica against clinical isolates of H. pylori. Component Mean [+ or -] SD * Essential oil 0.035 [+ or -] 0.13 (a) Thymol 0.043 [+ or -] 0.024 (a) Carvacrol 0.0181 [+ or -] 0.029 (b) Thymol + carvacrol *** 0.0175 [+ or -] 0.006 (b) * The different letters (a, b) indicate significant at the level [less than or equal to] 0.05 by Tukey's Range test. *** Equal to the combination of thymol and carvacrol in S. bachtiarica oil.