Toxicity of essential oil of Satureja khuzistanica: In vitro cytotoxicity and anti-microbial activity

Abstract

In nature, essential oils play an important role in the protection of the plants by exerting anti-bacterial, -viral, -fungal, -oxidative, -genotoxic, and free radical scavenging properties, as well as in some cases acting as insecticides. Several Satureja species are used in traditional medicine due to recognized therapeutic properties, namely anti-microbial and cytotoxic activities. The purpose of the present work was to determine the biologic activity of the essential oil of S. khuzistanica Jamzad (Lamiaceae) against four human cancer cell lines, as well as its inhibitory effects against a wide array (i.e. n = 11) of pathogenic bacteria and fungi. The essential oil was isolated by hydro-distillation and analyzed by GC-FID and GC-MS. Carvacrol (92.87%) and limonene (1.2%) were found to be the main components of the isolated oil. Anti-microbial activity of the essential oil was assessed using a disc diffusion method; an MTT cytotoxicity assay was employed to test effects of the oil on each cancer cell line. The oil exhibited considerable anti-microbial activity against the majority of the tested bacteria and fungi. The test oil also significantly reduced cell viability of Vero, SW480, MCF7, and JET 3 cells in a dose-dependent manner, with the IC₅₀ values calculated for each cell type being, respectively, 31.2, 62.5, 125, and 125 μg/ml. Based on the findings, it is concluded that the essential oil of S. khuzistanica and its major constituents have a potential for further use in anti-bacterial and anti-cancer applications, pending far more extensive testing of toxicities in normal (i.e. primary) cells.

• Carvacrol
• essential oil
• GC-MS
• MTT assay
• Satureja

Introduction

Essential oils—volatile, natural, complex compounds characterized by a strong odor—are formed by aromatic plants as secondary metabolites (Bakkali et al., 2008). The essential oils and their components have a wide range of applications in food flavoring, fragrances, and perfumes, but are also widely used in traditional ethno-medicine. Thus, considerable attention has been increasingly focused on the biological activities of essential oils (Unlu et al., 2010). In nature, these oils play an important role in the protection of the plants by acting as anti-bacterial, anti-viral, and anti-fungal agents, as well as functioning as insecticides and deterrents to the plant's consumption by herbivores. Some oils have also been shown to elicit anti-oxidative, anti-genotoxic, and free radical scavenging effects, as well as to exert overt cytotoxicity and induce apoptosis in cancer cells (Ahmad et al., 2012; Alpsoy et al., 2012; Goncalves et al., 2012; Mihajilov-Krstev et al., 2010; Ozkan et al., 2010; Tepe et al., 2005). The applications of essential oils in anti-cancer therapy may appear unconventional; however, their availability, pleasant aroma, and low or insignificant toxicity make them more attractive candidates for treatment of various chronic ailments (Sharma et al., 2010).

The genus Satureja L. (Lamiaceae) constitutes more than 200 species of aromatic herbs and shrubs widely distributed in Asia, the Mediterranean, and the Boreal region of North America. Fourteen species from this genus grow wild in Iran; nine species are endemic and distributed commonly in mountainous regions of the northern and western parts of the country (Jamzad, 1996; Mozaffarian, 1996; Rechinger, 1982). Many members of this genus have aromatic and medicinal properties. The leaves, stems, and flowers of some species are used for herbal teas and in local folk medicine to treat various diseases/maladies such as muscle pain, diarrhea, wounds, urinary tract infections, and gastroenteritis (Hadian et al., 2012).

The essential oils isolated from various species of Satureja have been shown to impart biological activities, e.g. anti-viral, anti-oxidant, anti-fungal, anti-bacterial, anti-diabetic, and anti-inflammatory (Abdollahi et al., 2003; Ciani et al., 2000; Eftekhar et al., 2009; Ghazanfari et al., 2006; Goren et al., 2004; Tzakou & Skaltsa, 2003). Those studies revealed that the Satureja species contain different amounts of biologically-active phenolic compounds, including thymol and carvacrol. Although the chemical composition (and anti-microbial activity) of essential oils of some Satureja species has been previously reported (Adiguze et al., 2006; Cavar et al., 2008; Ciani et al., 2000; Eftekhar et al., 2009), the anti-microbial and cytotoxic activity of oils from S. khuzistanica Jamzad, a plant that is endemic in southern Iran, have not been studied. This report presents results of studies to assess the cytotoxic activity of essential oils from S. khuzistanica, as well as their in vitro inhibitory effects against eight pathogenic Gram-positive/-negative bacteria and three pathogenic fungi.

Materials and methods

Plant material

The aerial parts of S. khuzistanica were collected from wild growing plants at full flowering stage in southern Iran. The plant material was first verified by Dr Ali Sonboli (Shahid Beheshti University, Tehran, Iran), and a voucher specimen then deposited at the Herbarium of Medicinal Plants and Drug Research Institute (MPH) at Shahid Beheshti University.

Essential oil isolation

The powdered plant aerial parts (250 g) were hydro-distilled using a Clevenger type apparatus for 3 h, according to the method recommended in the European Pharmacopoeia (Council of Europe, 1997). The resultant essential oil was dried over anhydrous sodium sulfate and stored at 4 °C until analyzed and tested. Measures of the final oil weight and volume were taken to calculate density of the stock. Dilutions of the oil in the various assays were then made from the parent oil stock by addition of dimethyl sulfoxide (0.1%; DMSO) to yield the desired final test concentrations.

Essential oil analysis and identification procedure

Gas chromatography-flame ionization detection (GC-FID) analyses of the oil were conducted using a Finnigan system (Thermoquest, Manchester, UK) equipped with a DB-5 fused silica column (60 m × 0.25 mm i.d., 0.25 µm film thickness; J&W Scientific, Folsom, CA). Nitrogen was used as the carrier gas at a constant flow of 1.1 ml/min. The split ratio was 1:50. The oven temperature was raised from 60 °C to 250 °C at a rate of 5 °C/min. Injector and detector (FID) temperatures were kept at 250 °C and 280 °C, respectively. GC-MS (mass spectroscopy) analysis was carried out in a Thermoquest Trace GC-MS instrument equipped with the same column and temperature programming as used for the GC analyses. The transfer line temperature was 250 °C. Helium was used as a carrier gas at a flow rate of 1.1 ml/min, with a split ratio of 1:50 (Hadian et al., 2012).

The constituents of the essential oils were identified by calculation of their retention indices under temperature-programmed conditions for n-alkanes (C₆–C₂₄) and the oil on a DB-5 column under the same conditions. Identification of individual compounds was made by comparison of their mass spectra with those in a reference mass spectra library (McLafferty, 2009) or with authentic compounds, and confirmed by comparison of retention indices with authentic compounds or those cited in the literature (Adams, 2001). Semi-quantitative data was obtained from FID area percentages without the use of correction factors.

Microbial strains

Eleven microbial strains were used in the anti-microbial activity assay. These included: Bacillus subtilis (ATCC 465), B. pumulis (PTCC 1274), Enterococcus faecalis (ATCC 29737), Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Pseudomonas aeruginosa (ATCC 85327), Aspergillus niger (ATCC 16404), Candida albicans (ATCC 10231), and Saccharomyces cerevisiae (ATCC 9763). All strains were obtained from the Pasture Institute (Tehran) and grown on appropriate agars as indicated below. The particular strains were selected for use here as they represent standard microbial strains that are major pathogens for humans.

Anti-microbial screening

The anti-microbial activity of the test oil (and separately its main component) was determined by the disk diffusion method outlined by the NCCLS (1997). Briefly, 0.1 ml of a suspension of a test micro-organism (10⁸ cells/ml) was spread over Mueller-Hinton Agar (bacteria) or Sabouraud Dextrose Agar (fungus) plates. Immediately after the spreading, a sterile 6 mm disk containing 10 µl (neat) of essential oil was placed in the center of the dish; this amount of oil was prepared so as to deliver 100 µg of the parent oil. Other plates/discs run in parallel contained either the major constituent (carvacrol) at a dose of 93 μg/disc, the standard anti-microbial agent ampicillin (at 10 μg/disc), or standard anti-fungal agent nystatine (at 30 μg/disc). The plates were incubated at 37 °C for 24 h for each bacteria and at 30 °C for 48 h for each fungi. Diameters of zones of inhibition were then measured and reported in millimeters. Triplicate tests were carried out in all experiments.

Determination of minimum inhibitory concentration (MIC)

MIC values were determined using a broth micro-dilution assay recommended by NCCLS (1999). Serial-fold dilutions of the essential oil (10.0–0.05 mg/ml) and separately, of the major constituent carvacrol, were made in Mueller-Hinton Broth containing 0.5% (v/v) Tween 80 (for bacterial assay) or in Sabouraud Dextrose Broth with 0.5% Tween 80 (for fungal assay). Fresh microbial suspensions prepared from cultures grown overnight in the same media were added to yield a final level of 5 × 10⁵ organisms/ml. Controls of medium with microorganisms or the essential oil/carvacrol alone were included. The inocula and test materials were then co-plated in wells of a microtiter plate, and then incubated (37 °C for 24 h for bacteria; 30 °C for 48 h for fungus). The first dilution demonstrating no microbial growth was recorded as the MIC.

Cytotoxicity against cancer cells

The human colon adenocarcinoma (SW480), breast adenocarcinoma (MCF7), and choriocarcinoma (JET 3) cell lines, as well as a monkey kidney cell line (Vero), were each obtained from the Pasteur Institute of Iran (Tehran, Iran). Cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY) and 1% penicillin-streptomycin, at 37 °C, in humidified air containing 5% CO₂. Cytotoxicity against each cancer cell line was assessed by a tetrazolium-based colorimetric assay (MTT) that measures reduction of tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) to formazan, mainly via activity of mitochondrial enzymes. In brief, 100 µl of cell suspension was plated at a density of 2 × 10⁴cells/well in a 96-well plate, and subsequently incubated at 37 °C in a 5% CO₂ humid incubator for 24 h. Thereafter, the essential oil at different concentrations was added to each well (triplicate wells per dose) and the incubation was continued for 24 and 48 h. The concentrations tested ranged from 7.8–1000 μg/ml; these values were selected based upon results from a previous study (Yousefzadi et al., 2009).

At each selected timepoint, l0 µl of MTT dye solution (5 mg/ml) was added to each well, and the plate incubated for a further 4 h at 37 °C. After removal of the MTT dye solution/medium, each well received 100 µl DMSO (0.1%) and the absorbance at 490 nm was then quantified using an ELISA plate reader. The extent of cytotoxicity induced was calculated by comparing absorbance values against those in control wells (cells treated only with 0.1% DMSO). Cytotoxicity was expressed as the concentration of oil inhibiting cell viability by 50% (IC₅₀). All tests and analyses were run in triplicate, and the means were calculated.

Statistics

All data are reported as mean ± SD. Analysis of variance was performed using SPSS 19.0 (IBM, SPSS) for Windows. Significant differences between means were determined using the Duncan’s multiple range test in ANOVA.

Results

Chemical composition of essential oil

The essential oil obtained by hydrodistillation of the aerial parts of S. khuzestanica was subjected to GC-FID and GC/MS to identify composition. Qualitative and quantitative analytical results are shown in Table 1; compounds are listed in order of their elution off the DB-5 column. The essential oil yield was 0.42% (w/w) on a dry weight basis. In this study, 19 compounds were identified; among these, carvacrol (92.87%) and limonene (1.2%) were the main compounds.

Table 1. Chemical composition of the essential oil of S. khuzistanica.

No	Component	RI	%	ID method
1	α-Thujene	925	0.21	RI, MS
2	α-Pinene	933	0.18	RI, MS, CO
3	Myrcene	981	0.16	RI, MS
4	α-Terpinene	1013	0.40	RI, MS, CO
5	p-Cymene	1017	0.51	RI, MS, CO
6	Limonene	1026	1.20	RI, MS, CO
7	Z-β-Ocimene	1036	0.18	RI, MS
8	γ-Terpinene	1053	0.52	RI, MS, CO
9	trans-Sabinene hydrate	1081	0.57	RI, MS
10	Terpin-4-ol	1163	0.27	RI, MS
11	α-Terpinole	1175	0.14	RI, MS
12	Thymol	1266	0.11	RI, MS, CO
13	Carvacrol	1282	92.87	RI, MS, CO
14	Thymyl acetate	1329	t	RI, MS
16	β-Caryophyllene	1425	0.40	RI, MS, CO
17	α-Humulene	1427	0.22	RI, MS
18	β-Bisabolene	1501	0.14	RI, MS
19	trans-β-Bisabolene	1522	t	RI, MS
	Total		98.08

RI, retention indices relative to C6–C24 n-alkanes on a DB-5 column; MS, mass spectroscopy; CO, co-injection with authentic compounds; t, trace, i.e. < 0.1% by MS, < 0.05% by CO.

Anti-bacterial and anti-fungal activity

Results of the evaluation of the anti-microbial properties of the S. khuzistanica essential oil—using a disk diffusion method and minimum inhibition concentration (MIC)—are shown in Tables 2 and 3. Inhibition zones (IZ) and MIC values of the essential oil and its major constituent carvacrol showed a variability of inhibition among the bacteria and fungi tested. The results indicated that the oil exhibited strong anti-microbial activity and moderate anti-fungal activity, except for A. niger. The terms strong, moderate, etc. to describe activity were derived from a scaling method reported by Baron & Finegold (1990), wherein IZ zones were used to reflect potency of a test agent, i.e. weak (< 7), moderately active (7–14); highly active (> 14).

Table 2. Anti-microbial activity of essential oils from S. khuzistanica.

Micro-organisms	S. khuzistanica^a		Carvacrol^a		Ampicillin^a		Nystatine^a
	IZ^b	IZ [mm]/µg	IZ	IZ [mm]/µg	IZ	IZ [mm]/µg	IZ	IZ [mm]/µg
B. subtilis	42.0 ± 0.2	0.420	43.0 ± 0.4	0.462	14.0 ± 0.4	1.400
B. pumulis	43.0 ± 0.5	0.430	42.0 ± 0.1	0.452	15.0 ± 0.3	1.500
E. faecalis	20.0 ± 0.2	0.200	22.0 ± 0.7	0.237	11.0 ± 0.3	1.100
S. aureus	30.0 ± 0.9	0.300	30.0 ± 0.2	0.323	13.0 ± 0.3	1.300
S. epidermidis	45.0 ± 0.5	0.450	49.0 ± 0.4	0.527	19.0 ± 0.5	1.900
E. coli	39.0 ± 0.3	0.390	38.0 ± 0.2	0.409	12.0 ± 0.2	1.200
K. pneumoniae	19.0 ± 0.7	0.190	23.0 ± 0.6	0.247	–	–
P. aeruginosa	11.0 ± 0.5	0.110	12.0 ± 0.8	0.129	10.0 ± 0.3	1.000
A. niger	0	–	9.0 ± 0.7	0.096			16.0 ± 0.4	0.530
C. albicans	24.0 ± 0.5	0.240	23.0 ± 0.9	0.247			18.0 ± 0.5	0.600
S. cerevisiae	31.0 ± 0.6	0.310	32.0 ± 0.6	0.344			18.0 ± 0.2	0.600

Results (shown as mean ± SD) were obtained from three independent experiments, each performed in duplicate.

^aTested at 100 µg oil/disc, 93 µg carvacrol/disc, 10 μg ampicillin/disc, and 30 μg nystatine/disc.

^bInhibition Zone includes diameter of disc (6 mm).

– = Inactive.

Table 3. Anti-microbial activity of essential oils from S. khuzistanica.

Micro-organisms	S. khuzistanica MIC^a	Carvacrol MIC
B. subtilis	0.23	0.23
B. pumulis	0.23	0.23
E. faecalis	3.75	3.75
S. aureus	0.93	0.46
S. epidermidis	0.23	0.23
E. coli	0.46	0.46
K. pneumoniae	3.75	1.87
P. aeruginosa	15.00	15.00
A. niger	–	15.00
C. albicans	1.87	1.87
S. cerevisiae	0.93	0.46

Results (shown as mean ± SD) were obtained from three independent experiments, each performed in duplicate.

^aMinimum inhibitory concentration values as mg/ml.

– = Inactive.

The essential oil was quite effective against growth of Gram-positive Staphylococcus and Bacillus and Gram-negative E. coli. The data showed that S. epidermidis, B. pumulis, and B. subtilis were the most sensitive of the micro-organisms tested, with IZ values of 45 [±0.5], 43 [±0.5], and 42 [±0.2] mm (Table 2); each of the organisms yielded an MIC value of 0.23 mg/ml (Table 3) as well. Of the test pathogens, P. aeruginosa, K. pneumonia, and E. faecalis were the most resistant against effects from the oil. Evaluation of anti-microbial activity of the main constituent of the oil revealed that the inhibitory activity with carvacrol was comparable to that of parent oil (Tables 2 and 3). In comparison, the IZ values for the standard anti-microbial ampicillin ranged from 12–19 overall (and there was no IZ against K. pneumoniae). On a per-concentration basis, compared against the ampicillin, the oil showed a lower effect than that of the standard. Specifically, inhibition zones ranged from 0.11–0.45 mm/µg oil, while the ampicillin had a comparative effect of 1.00–1.90 mm/µg. Depending on the organism, this translates to a lesser activity of 1.5–10-fold for the oil compared to the ampicillin standard.

Analysis of anti-fungal activity revealed that A. niger was absolutely insensitive to the oil (Table 2). In contrast, S. cervisiae and C. albicans were very sensitive, with inhibition zones of 31 [±0.6] and 24 [±0.5] mm; each of the organisms gave rise to an MIC value of, respectively, 0.93 and 1.87 mg/ml (Table 3). Interestingly, while the ability of the intact oil in preventing growth of these two specific organisms was comparable to that by the major constituent carvacrol, the carvacrol alone exhibited inhibitory effects against the A. niger. In comparison to the oil and the carvacrol alone, IZ values for the standard anti-fungal nystatine ranged from 16–18 overall across all three fungi tested. On a per-concentration basis, compared against the nystatine standard, the oil showed similar effects produced by the standards. On a per-concentration basis, compared against the nystatine, the oil (and carvacrol alone) had a lower effect than the standard. Specifically, inhibition zones ranged from 0.24–0.31 mm/µg oil, while the nystatine had a comparative effect of ≈0.6 mm/µg. Depending on the organism, this translates to a lesser activity of ≈2-fold for the oil compared to the nystatine standard.

Cytotoxicity against cancer cells

To evaluate the cytotoxic effect of the test essential oil against commonly used cancer cell lines (human and monkey), assays were performed using MTT reduction as the critical end-point. Growth of all four cell lines, i.e. human colon adenocarcinoma (SW480), breast adenocarcinoma (MCF7), and choriocarcinoma (JET 3), as well as monkey kidney (Vero) cells, was inhibited in a dose-related manner after 24 or 48 h of exposure to the essential oil (Figure 1). IC₅₀ values were estimated to be 31.2, 62.5, 125, and 125 μg oil/ml for the Vero, SW480, MCF7, and JET 3 cells, respectively.

Figure 1. Cytotoxic effect of S. khuzistanica essential oil in JET 3, MCF7, Vero, and SW480 cells evaluated using an MTT assay. Results are presented as viability ratio compared to the control group (treated with DMSO). Values expressed as mean (±SD) of five independent experiments. *p <  0.05 and **p <  0.01; significantly different from the solvent (‘0’) control.

Discussion

In this study, it was demonstrated that S. khuzistanica essential oil has significant anti-microbial activity. In addition, a cytotoxic effect of the oil against cancerous cell lines was noted. This S. khuzistanica oil was characterized by a high amount of carvacrol (92.87%). Hadian et al. (2012) recently characterized the essential oil of S. khuzistanica; that group noted a total of 19 compounds, comprising 98.3% of the total oil composition. In those samples, monoterpenoids (94.1%)—with carvacrol (68.8%) and its precursors p-cymene (7.3%) and γ-terpinene (5.5%)—were the principal components. In another study, the oil of S. khuzistanica was characterized by high amounts of p-cymene (39.6%), carvacrol (29.0%), and γ-terpinene (18.9%) (Sefidkon & Ahmadi, 2000). Here, we noted varieties containing high levels of carvacrol. Carvacrol, p-cymene, and especially thymol are usually present as major compounds in the oils of other Satureja species (Gohari et al., 2005; Sefidkon & Jamzad, 2005; Sonboli et al., 2004). The potential reasons our results differ from the aforementioned other studies might be related to genetic diversity, climatic conditions, and/or ecological differences (Runyoro et al., 2010).

By comparing the anti-microbial activity of the essential oil of S. khuzistanica in this study and its major compound, it can be suggested that carvacrol is the responsible agent. The results also showed that carvacrol alone exhibited an anti-bacterial activity that was greater than its anti-fungal activity. This would be in keeping with the finding here that the essential oil of S. khuzistanica had both anti-fungal and anti-bacterial activity, but IZ and MIC values with bacteria were higher than those with the fungi. Previously, anti-microbial activities of essential oils from Satureja species have been reported, although variable in degree and spectrum of activity—according to the exact plant species and related composition. Eftekhar et al. (2009) showed anti-microbial activity of S. spicigera essential oil against six bacterial strains; these authors claimed that the high amounts of carvacrol and thymol (∼89.8%) were responsible for the inhibitory effects. This is not surprising in that the analyses of the anti-microbial activities of essential oils from four Satureja species (analyzed against phyto-pathogenic bacteria Erwinia amylovora) showed that phenolic compounds like carvacrol, mono-terpene limonene, and the alcohols linalool and borneol were mostly responsible for high anti-microbial activity (Mihajilov-Krstev et al., 2010). It has been proposed that essential oils rich in phenolic compounds like carvacrol, p-cymene, γ-terpinene, or thymol exert strong anti-microbial activities (Oke et al., 2009).

While the oil and its major constituent carvacrol did display notable anti-bacterial and anti-fungal effects in the assays here, it is clear that each is not as effective as their ‘gold’ standard (ampicillin and nystatine, respectively) counterparts. This could be due to the fact that as a β-lactam antibiotic, ampicillin penetrates the outer membrane of (primarily Gram-negative) bacteria via porins (James et al., 2009) and then acts as a competitive inhibitor of transpeptidase needed by the bacterium to make a cell wall, an effect that ultimately leads to cell lysis (at the extreme end) and an overall reduced ability for the pathogens to successfully replicate. As with other anti-fungals like natamycin or amphotericin B, nystatine binds ergosterol on the fungus cell membrane. At sufficient nystatine concentrations in the membrane, this allows for the formation of pores that, in turn, permit ions (i.e. K⁺) to leak from the cell and, ultimately, cause cell death. In contrast, carvacrol (and, hence, the parent oil here) is thought to impart anti-microbial effects primarily via induction of changes in the cell membranes of the targeted organisms, with shifts in the profiles of unsaturated fatty acids that lead to alterations in the cell envelope and ultimately cell death due, in part, to impaired regulation of osmolarity (di Pasqua et al., 2007). Thus, a strong dependence for ‘preferred’ pathogen cell wall compositions (i.e. preferential degree/profiles of the fatty acids present) vs the relative availability of porins (or ergosterol) in/on a target pathogen presents a key means by which we might explain the differences seen here in effects (on a per-concentration basis) between the oil/carvacrol and the ampicillin/nystatine standards.

In this study, the S. khuzistanica essential oil demonstrated cytotoxic activity against various types of cancer cells. More studies are imperative to explore the potential application of this material for pharmaceutical purposes as well as to better characterize the mechanisms of the activities observed here. To date, the cytotoxic activity of S. khuzistanica essential oil has not been studied. From the data presented here, we hypothesize that, because carvacrol accounts for greater than 93% of the composition of the oil and, as noted above, the levels of anti-microbial activity for carvacrol alone are nearly equivalent to that induced by the intact oil, the carvacrol may also account for the overall cytotoxicity against the cancer cell lines tested.

It should be noted that the contributing effects of the other constituents can not be ruled out at this time; as noted by Burt (2004), many studies have shown that minor components play a role in anti-bacterial activity, possibly by producing synergistic effects with other components (Burt, 2004). It is equally likely to be the case in cytotoxic activities against transformed cells. Further studies are clearly 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.

Conclusions

The results of this study clearly indicate that the essential oil of S. khuzistanica is a strong anti-microbial mixture that also possesses cytotoxic activities against various types of transformed cells. With respect to cancer, the cytotoxic properties of this oil at low concentrations may eventually support an applicability of the oil in combating cancer, with a hopefully minimum amount of adverse effects toward normal healthy cells. Further toxicological studies in normal primary cells are clearly needed to validate this potential.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of this paper.

This work was supported by the Institute of Science and High technology and Environmental Sciences, Graduate University of Advanced Technology, Kerman, Iran [Grant number 1/2955].