Gas chromatography-mass spectrometry analysis

The analysis of the essential oil was performed using a Trace MS Thermoquest-Finnigan, chromatograph equipped with a fused silica capillary DB-5 column (60 m×0.25 mm i.d., film thickness 0.25 µm). An electron ionization system, IE: 70 eV, was used for GC-MS detection. The carrier gas was helium at a flow rate of 1.1 ml/min. Injector and detector temperatures were set at 250 and 300 ºC, respectively. The oven temperature program was 60-250 ºC at the rate of 5 ºC/min and finally held isothermally for 10 min. Diluted samples (1/100, v/v, in dichloromethane) of 0.2µl were injected manually in the splitless mode. Identification of components of the essential oil was based on GC retention indices and computer matching with the Wiley and Adams libraries, as well as by comparison of the fragmentation patterns of the mass spectra with those reported in the literature.

Plant material

All plant material of S. khuzistanica and S. rechingeri was collected from the Pol-e Dokhtar of Lorestan and Zarrinabad region of Ilam Province, Iran, respectively. The voucher specimen (No.: 11893 for S. khuzistanica and 13228 for S. rechingeri) has been deposited in the Herbarium of the Agricultural Research Center of Lorestan, Khorramabad, Iran.

Essential oil Isolation

The essential oils of powdered aerial parts of S. khuzistanica (250 g) and S. rechingeri (200 g) were obtained by using a Clevenger apparatus with hydrodistillation method for 3 h, giving yellow oil in 0.9 % and 0.5 % (v/w) yields, respectively. The oil was dried with anhydrous Sodium Sulphate and stored under nitrogen.

ChE assay

Human cholinesterase activity measurements were carried out by a modified Ellman’s method [³¹]. The reaction mixtures for the determination of IC₅₀ values, the median inhibitory concentration, consisted of (5,5′- dithiobis(2-nitrobenzoic acid (DTNB: 10⁻⁴ M), 50 ml; inhibitor, × μl (5-250); acetylthiocholine iodide (ATCh) (1.35×10⁻⁴ M), 30 mL; 70mM phosphate buffer (Na₂HPO₄/NaH₂PO₄, pH = 7.4) (885-x) μl, and hAChE (diluted 100 times in phosphate buffer solution, 30 μl). The absorbance change was monitored at 412 nm for 3 min at 37 °C with three replicates in each experiment. In the absence of an inhibitor, the absorbance change was directly proportional to the enzyme level. The plot of V_I/V₀ against log [I] (where V_I and V₀ are the activity of the enzyme in the presence and absence of inhibitors, respectively, and [I] is the inhibitor concentration), gave the IC₅₀ values of samples. Similarly, the activity of BChE was determined by measuring thiocholine which reacted with DTNB after hydrolysis of BTCh. The lyophilized BChE was diluted with 100 mM phosphate buffer (pH = 8) for use in the activity assay.

Docking studies

The initial coordinates of the ChE enzyme were taken from RCSB Protein Data Bank (PDB code: 4EY6 for AChE and 4BDS for BChE), which are complex with GNT and THA inhibitors, respectively. All the crystal water molecules and bound inhibitors were removed from the PDB file. The structure of Carvacrol was used for the docking study, which was carried out through Autodock Vina v.1.1.2 [³²]. Required PDBQT files for the receptor protein and ligand molecules were prepared by Auto Dock Tools v.1.5.6 [³³]. The receptor was kept rigid, and ligands were permitted to be flexible. During PDBQT file preparation, polar hydrogen atoms and Kollman united atom partial charges were added to the protein atoms (ChE) [³⁴]. A docking grid box with sizes (X: 20, Y: 20, Z: 20) was built with 50, 50, and 50 points in the catalytic site of the protein, and the center of the box was set in the centroid of the bound inhibitors in the PDB file (GNT [X: -9.94, Y: -43.49, Z: 30.29 for chain A and X: 8.68, Y: -60.48, Z: -24.27 for chain B] in AChE and THA [X: 132.99, Y: 116.01, Z: 41.21] in BChE). Docking results were clustered with a root mean square deviation (RMSD) of 0.85Å and evaluated using the Pymol software.

Cytotoxicity assay

The cytotoxic properties of the essential oil of S. khuzistanica and S. rechingeri were determined on two human cancer cell lines: ovarian A2780 and prostate PC-3 using doxorubicin as a reference drug. Briefly, cell lines were grown in RPMI-1640 containing 10 % FBS at 37 ºC in a humidified atmosphere of 5 % CO₂/95 % air and were seeded at a density of 2×10³-8×10³ cells per well in a 96-well plate. Samples were dissolved in the culture medium to give various concentrations. After incubation for 24 h, the resultant solutions were subsequently added to a set of wells. The microtiter plates were incubated at 37 ºC in a humidified atmosphere of 5 % CO₂/95 % air for 24 and 48 h after adding all samples. Cytotoxic screening by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed. The MTT solution (10 µL, 5 mg/mL) was added to into each well at the end of each incubation period and the cultures were incubated for 3-4 h. After the removal of the supernatant, DMSO (200 µL) was added to dissolve the formazan crystals. The absorbance was measured at 540 nm on an Eliza reader. The cytotoxicity was evaluated based on the percentage of cell survival in a dose-dependent manner relative to the negative control. The IC₅₀ value is defined as a sample concentration that inhibits 50 % of the cell growth.

Statistical analysis

Samples were analyzed in triplicate and a one-way analysis of variance was performed using SPSS version 10. Significant differences were detected at P<0.05. All experiments were carried out in triplicate. Data are expressed as mean ± standard deviation.

Chemical evaluation

The constituents of the essential oil of Satureja khuzistanica and Satureja rechingeri identified by GC-MS analysis and the quantitative data are summarized in Fig. 1 and Table 1. Essential oil from Satureja khuzistanica indicated the presence of thirty-seven components, whereas, essential oil of Satureja rechingeri showed the presence of thirty-eight compounds, representing 98.09 % and 99.46 % of the total identified ingredients. Interestingly, twenty-eight components were generally found in both essential oils. Among them, Carvacrol and Thymol were found the most abundant component in S. khuzistanica and S. rechingeri essential oils (68.7 % and 51.28 %, respectively). Other major ingredients present in S. khuzistanica essential oil were p-Cymene (4.62 %), 4- Terpineol (3.16 %), and γ-Terpinene (2.94 %), while in S. rechingeri essential oil, Carvacrol (22.08 %), Linalool (4.37 %), cis-β-Bisabolene (3.08 %), cis-Sabinene hydrate (3.01 %), 4-Terpineol (2.99 %), Caryophyllene oxide (2.07 %), and p-Cymene (2.06 %) were the principal components (Table 1). The oil obtained from S. khuzistanica in this study was found to contain more chemical ingredients than the essential oil of this spice in all reported studies with the hydrodistillation method [²⁹, ³⁵, ³⁶]. For instance, 21 compounds in the essential oil of the cultivated S. khuzistanica and 12 compounds in the essential oil of its wild spice were detected [³⁵].

Fig. 1 GC-MS chromatogram of essential oil of (a) S. khuzistanica and (b) S. rechingeri. 

On the other hand, the essential oil of S. rechingeri in the present study was found to contain less/more chemical compounds than the essential oil of this spice in the reported studies with the hydrodistillation method. For example, Alizadeh has reported essential oil compositions of S. rechingeri at different phenological stages to contain forty-seven components [²⁶]. In another study, fifty-three constituents were identified in the essential oil of S.rechingeri at the beginning of the flowering stage by Sefidkon et al., while twenty-three compounds were detected in the oil obtained through hydrodistillation at the full flowering stage [³⁷].

A literature review has also shown variations between chemical compositions of title two Satureja species oils. For instance, Farzaneh et al. reported Carvacrol (48 %), p-Cymene (18.5 %), and γ-Terpinene (11 %) as the main components of S. khuzistanica essential oil growing in Lorestan, which was in agreement with results of this study [³⁸]. In a study conducted by Yousefzad et al, Carvacrol (92.87 %) and limonene (1.2 %) were obtained as major components in S. khuzistanica oil [²⁹]. Carvacrol (82.5%), γ-Terpinene (2.7 %), and p-Cymene (2.6 %) were also obtained as dominant compounds in the essential oil of S. rechingeri grown in the Dehloran region of Ilam Province [³⁹]. As shown in Table 1, while amounts of Carvacrol, p-Cymene, 4- Terpineol, and γ-Terpinene significantly decreased in S. rechingeri oil, increases in the amount of these components were observed for the essential oil of S. khuzistanica. Further, a decrease in the amount of Linalool, cis-β-Bisabolene, cis-Sabinene hydrate, and Caryophyllene oxide were observed for the extracted oil obtained from S. khuzistanica whereas, the value of these components increased in S. rechingeri extract oil. Numerous studies have characterized Carvacrol as the most abundant constituent in the essential oils obtained from S.khuzistanica and S.rechingeri [⁴⁰], whereas Carvacrol and Thymol were identified as the dominant compounds in the essential oils extracted from S.khuzistanica and S.rechingeri in this study, respectively. The chemical ingredient of extract oils may change from plant to plant even in the same species.

Molecular docking studies

The essential oils studied herein were found to be rich in Carvacrol, and considering the interesting IC₅₀ values displayed by Carvacrol on AChE and BChE (Table 2), the docking studies were executed at the active site 3D space of both enzymes to get an insight of the intermolecular interactions of the title component. The binding models of Carvacrol against AChE and BChE are depicted in Figures 3(a), S1, and 3(b). The docking calculations of Carvacrol at the active site gorge of AChE and BChE revealed that the best conformation of this compound bound to both enzymes with a binding affinity (∆G) from -6.7 to -6.4 Kcal/mol. The ∆G for GNT bound inhibitor in complex with 4EY6 were obtained -10.3 Kcal/mol for Chain A and -10.1 Kcal/mol for Chain B, and as well as those of THA bound inhibitor in complex with 4BDS were obtained -8.3 Kcal/mol, which is in agreement with the in vitro results listed in Table 2. Carvacrol fits in the hydrolase catalytic pocket of the X-ray crystal structure of acetylcholinesterase and the hydroxyl group of Carvacrol can establish strong or weak hydrogen bonds with the important amino acid residues of AChE. In chain A (Fig. 3(a)), the O-H group of Carvacrol formed two hydrogen bonds with N (length = 2.56 Ǻ) and O (3.12 Ǻ) of Arg296, and a hydrogen bond with the N (2.51 Ǻ) atom of Phe295. In chain B (Fig. S1), the hydroxyl group of the same compound showed hydrogen bonding to the important residues of AChE, as this group showed hydrogen bonds with the two oxygen atoms of the Asp74 (3.21 Ǻ) and Tyr124 (3.38 Ǻ). Additionally, interactions C-H…π could be formed between isopropyl and methyl moiety of Carvacrol with Tyr341 (3.10 Ǻ in chain A; 2.93 Ǻ in chain B) and Trp286 (3.58 Ǻ in chain A; 3.61 Ǻ in chain B), which are some of the eminent residues of AChE. The lowest energy conformer of Carvacrol showed at least four hydrogen bonds at the active site of BChE (Fig. 3 (b)). Group O-H of Carvacrol formed hydrogen bonds with OE1 (3.28 Ǻ) and OE2 (2.68 Ǻ) atoms of Glu197 and with N (3.27 Ǻ) atom of Gly439, and this group showed hydrogen bond with the O (3.57 Ǻ) atom of His438. In addition, the phenyl ring of Carvacrol is involved in π...π stacking interaction with Trp82 and His438 of the catalytic pocket. Comparing the docking results and the in vitro binding data reveals that the higher inhibitory effect of the S. khuzistanica oil in comparison to the S. rechingeri is strongly influenced by the presence of more Carvacrol percentage and all the titled interactions.

Fig. 3 Docking of Carvacrol in AChE (PDB ID: 4EY6) (Chain A (a)) and BChE (PDB ID: 4BDS) (b). AChE and BChE are represented in green and Carvacrol in orange, (hydrogen bonds between O-H…N (Arg296), O- H…N (Phe295), and C-H…π interaction (Tyr341) (a); O-H…O (Glu197), O-H…N (Gly439), O-H…O (His438), and π-π stacking interaction (Trp82) (b). 

Anticancer activity

Due to the significant side effects of the synthesized anticancer drugs, researchers have been focused on the use of natural products for cancer therapy [⁵⁰]. There are more than one thousand herbs that have been noticed to have remarkable anticancer potentials. Especially the essential oils of the plant parts have been studied and applications of them in cancer therapy reported so far. Further, it is anticipated that they induce lesser adverse effects in proportion to synthetic medications [⁵¹]. To the best of our knowledge, little research about anticancer properties of essential oil of Satureja species has been reported [⁵²], and this study is the first report on the in vitro cytotoxicity of S. khuzistanica and S. rechingeri essential oil against human ovarian A2780 and human prostate PC-3 cancer cell lines. Cell culture treatments were assessed following different concentrations of the samples for two different times of exposure (24 h and 48 h). The selected samples have a growth-inhibitory effect on the cancer cells in a concentration and time-dependent pattern (Figures 4(a-d)), and the IC₅₀ values are reported in Table 3.

Fig. 4 Cytotoxic effects of essential oil of S. khuzistanica and S. rechingeri in different concentrations and times against A2780 and PC-3 cell lines (a - d). The data were expressed as the mean ± SD from 3 independent experiments (P<0.05). 

The standard anticancer drug, doxorubicin, was also taken as control [⁵³]. Consequently, a comparison of the IC₅₀ values revealed that by increasing the incubation time from 24h to 48h, a slight decrease in IC₅₀ value was found in the A2780 cell line (from 1160.30±5.11 to 1028.19±4.54 µg/mL) for S. khuzistanica essential oil, and a significant reduction was observed against PC-3 cell line for the same sample from 987.05±3.73 to 832.17±3.34 µg/mL. In the case of S. rechingeri essential oil, the IC₅₀ values show a considerable reduction from 1077.58±5.94 to 488.96±3.19 µg/mL against the A2780 cell line, and from 1431.19±6.25 to 767.22±3.19 µg/ml on the PC-3 cell line. Then, the essential oil of S. rechingeri (IC₅₀: 488.96±3.19 µg/ml) was revealed higher activity than the essential oil of S. khuzistanica (IC₅₀: 1028.19±4.54 µg/mL) against A2780 cell line. Also, for the PC-3 tumor cell line, S. rechingeri essential oil (IC₅₀: 767.22±3.19 µg/mL) display a slight increase over the essential oil of S. khuzistanica (IC₅₀: 832.17±3.34 µg/mL).

A literature review also presents that Carvacrol and Thymol are effective ingredients for the inhibition of cancer cells with relatively very low cytotoxic activity on normal cells [⁵⁴-⁵⁶]. Besides, numerous studies have indicated that minor compounds play a role in antibacterial activity, possibly by producing synergistic effects with other constituents [⁵⁷]. It is equally likely to be the case in cytotoxic activities on transformed cells. Hence, further research are warranted to assess the cytotoxic potentials of the more minor constituents alone and then in various combinations to truly discern their role(s) in the toxicity against transformed cells. To study the degree of selectivity in the cytotoxic activity of the samples, assays using lymphocyte isolation from whole human blood were carried out on the selected samples. Selectivity assay showed that survival values were from 89 % to 93 % for these samples.