Bioactivities of black cumin essential oil and its main terpenes from Tunisia

Ex vivo antioxidant, anti-inflammatory, anticancer and antibacterial activities of the essential oil from Tunisian Nigella sativa seeds and its main terpenes (p-cymene, γ-terpinene, thymoquinone, β-pinene, carvacrol, terpinen-4-ol and longifolene) were determined. The essential oil exhibited strong ex vivo antioxidant activity, inhibiting DCFH oxidation with an IC50 of 1.0 μg/ml, and high anti-inflammatory activity, inhibiting NO radical excretion with an IC50 value of 6.3 μg/ml. Thymoquinone was found to be the most active to decrease DCFH oxidation and NO excretion. The oil was found to significantly inhibit the growth of A-549 and DLD-1 cancer cell lines (IC50 values of 43.0 and 46.0 μg/ml, respectively) and to exert antibacterial activity against Staphylococcus aureus and Escherichia coli with IC50 values of 12.0 and 62.0 μg/ml. The anticancer and antibacterial activities could be mainly due to the action of thymoquinone and longifolene. © 2009 SAAB. Published by Elsevier B.V. All rights reserved.


Introduction
Nigella sativa L. (Ranunculaceae), commonly called black cumin and "sinouj" in Tunisia, is an annual herbaceous plant cultivated in different parts of the world, mainly in countries bordering the Mediterranean Sea. The seeds are used extensively for flavouring and medicinal purposes. They are used in the preparation of a traditional sweet dish and eaten with honey and syrup and for sprinkling on bread, flavouring of foods (as a spice) , especially bakery products and cheese (Takruri and Dameh, 1998). The seeds are also used in traditional medicine as a natural remedy for several illnesses that include asthma, hypertension, diabetes, inflammation, cough, bronchitis, headache, eczema, fever, dizziness and influenza, and as a carminative, diuretic, lactagogue and vermifuge (Ali and Blunden, 2003). Extensive studies have been conducted on the pharmacological properties of solvent extracts of N. sativa seeds including ethanol (Swamy and Tan, 2000;Kumara and Huat, 2001), methanol (Al-Naggar et al., 2003;Thippeswamy and Naidu, 2005), aqueous (Al-Ghamdi, 2001;Thabrew et al., 2005) and diethyl ether extracts (Hanafy and Hatem, 1991;Aljabre et al., 2005), while there are some reports on the biological activities of the essential oil (Burits and Bucar, 2000;Islam et al., 2004;Nair et al., 2005;Mbarek et al., 2007).
The biological properties of the essential oils have been found to be directly linked to their chemical compositions, which are influenced by the origin of the plants (Celiktas et al., 2007). A variety of chemotypes have been described in the literature. An Iranian N. sativa essential oil was found to be dominated by phenylpropanoid components and displayed a trans-anethole chemotype (Nickavar et al., 2003) while a chemotype with 33% p-cymene and 26.8% thymol and the preponderance of monoterpenes was reported for N. sativa essential oil from Morocco (Moretti et al., 2004). In addition, Benkaci- Ali et al. (2007) demonstrated a considerable difference in the chemical composition of the Algerian N. sativa seed essential oils obtained from specimens growing in two localities; the oil being dominated by both alcohols and acids or by monoterpene hydrocarbons, according to the origin.
The objective of this study was to investigate, for the first time, the ex vivo antioxidant activity as well as the anticancer, anti-inflammatory and antibacterial activities of Tunisian N. sativa essential oil. Moreover, in order to identify bioactive volatile components, the biological activities of the main, commercially available volatiles present in the oil, were simultaneously evaluated.

Plant material and essential oil extraction
Seeds of N. sativa were collected at maturity, in July 2006, from cultivated plants from the region of Menzel Temime (Northeastern Tunisia). The specimen was authenticated by Dr. Abedrrazak Smawy at Biotechnologic Center, Technopark of Borj-Cédria, and the voucher specimen "RNS18" was deposited in the herbarium of the Unit of Aromatic and Medicinal Plants at the Biotechnologic Center. The essential oil was obtained from seeds (100 g) by hydrodistillation for 90 min in a Clevenger type apparatus. The oil was then dried with anhydrous sodium sulphate and stored at − 20°C in darkness until used.

Analysis conditions
The essential oil composition was determined using gas chromatography with flame ionisation detection (GC-FID) and verified using GC with mass spectrometry (GC-MS) detection. For GC-FID, an Agilent 6890 GC, equipped with a polar Supelcowax-10 column (30 m × 0.25 mm × 0.25 µm) and an apolar DB-5 column with the same dimensions, was used. Helium was the carrier gas at a flow rate of 1 ml/min. A split ratio of 50:1 and injector and detector temperatures of 200°C and 260°C, respectively, were used. Initially, the oven temperature was set at 40°C for 2 min, then raised by 2°C/min to a final temperature of 210°C (held for 33 min). A Hewlett-Packard (Model HP5890) gas chromatograph coupled to a Model HP5972 mass spectrometer was employed for GC-MS analysis. Separation was achieved on the same DB-5 column, using identical oven temperature conditions. The ionisation energy was set at 70 ev. Conditions used for the carrier gas and the split ratio were the same as for GC-FID. However, the injector and detector temperatures were set at 225°C and 280°C, respectively. Identification of volatile constituents was made on the basis of their retention indices on both columns (Kovats, 1965) and their mass spectra, which were compared with reference data (Adams, 2007).

Antioxidant activity
Antioxidant activity was evaluated using the 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) assay as described by Girard-Lalancette et al. (2009), with some modifications. Briefly, WS-1 cells were plated in 96-wells microplates at 10,000 cells per well and incubated for 24 h at 37°C under 5% CO 2 . The cells were washed with 150 µl Hank's balanced salt solution (HBSS) at pH 7.4 and incubated for 30 min with 100 µl HBSS (pH 7.4) containing 5 µM DCFH-DA. The cells were then washed again with 150 µl HBSS. To assess the antioxidant activity, the cells were incubated either with increasing concentrations of essential oil or increasing concentrations of pure compounds in DMSO, in the absence or the presence of 200 µM tert-butylhydroperoxide (t-BuOOH). Fluorescence was measured immediately after t-BuOOH administration and again 90 min later, using an automated 96-well Fluoroskan Ascent Fl™ plate reader (Fluoroskan Ascent Fl, Thermo-Labsystems) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

Anti-inflammatory activity
To investigate the anti-inflammatory activity of N. sativa seed essential oil, nitric oxide (NO) production in LPS-stimulated RAW 264.7 cells was examined. Exponentially growing macrophages were plated in 24-well microplates (BD Falcon) at a density of 2 × 10 5 cells per well in 400 µl of culture medium and were allowed to adhere for 24 h at 37°C under 5% CO 2 . Cells were then treated with increasing concentrations of essential oil and pure compounds dissolved in DMSO. The final concentration of solvent in the culture medium was maintained at 0.5% (v/v) to avoid solvent toxicity. Cells were then stimulated with 100 µg/ml lipopolysaccharide (LPS) and incubated at 37°C under 5% CO 2 . After 24 h, cell-free supernatants were collected and nitrite production was measured using the modified method of Green et al. (1990). Griess reagent (50 µl of 1% sulphanilamide and 50 µl of 0.1% N-1-naphtylethylenediamine dihydrochloride in 2.5% H 3 PO 4 ) was added in equal volume (100 µl) to cell supernatant and incubated at room temperature for 30 min. N(G)nitro-L-arginine methyl ester (L-NAME) was used as a positive control. The absorbance at 540 nm was then measured using an automated 96-well Varioskan Ascent plate reader (Thermo Electron) and nitrite was quantified by comparison with a NaNO 2 standard curve.

Anticancer activity
Exponentially growing cells were plated at a density of 5 × 10 3 cells per well, in 96-well microplates (Costar, Corning Inc.) into 100 µl of culture medium and were allowed to adhere for 24 h at 37°C under 5% CO 2 before treatment. Then, 100 µl of increasing concentrations of essential oil or pure compounds in DMSO were added. The final concentration of solvent in the culture medium was maintained at 0.5% (v/v) to avoid solvent toxicity. The cells were incubated for 48 h in the presence or absence of essential oil or compounds. The cytotoxicity was assessed using the resazurin reduction test as described by O'Brien et al. (2000). Fluorescence was measured using an automated 96-well Fluoroskan Ascent Fl™ plate reader (Thermo-Labsystems) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Cytotoxic activity was expressed as the concentration of essential oil or compound inhibiting cell growth by 50% (IC 50 ).

Antibacterial activity
Antibacterial activity was assessed according to the microdilution method described by Banfi et al. (2003). Briefly, exponentially growing bacteria were plated in 96-well round bottom microplates (Costar, Corning Inc.) at a density of 25 × 10 3 gram-positive Staphylococcus aureus (ATCC 25923) or 5 × 10 3 gram-negative Escherichia coli (ATCC 25922) per well in 50 µl nutrient broth (Difco). Then, 100 µl of increasing concentrations of essential oil or pure compounds were added per well. The final concentration of solvent in the culture medium was maintained at 0.1% (v/v) to avoid solvent toxicity. Antibacterial activity was assessed by adding 50 µl of 4% resazurin to each well; the microplates were then incubated at 37°C. Fluorescence was measured after 6 h on an automated 96well Fluoroskan Ascent Fl TM plate reader (Thermo-Labsystems) using excitation and emission wavelengths of 530 nm and 590 nm, respectively. Antibacterial activity was expressed as the concentration of essential oil or compound inhibiting bacterial growth by 50% (IC 50 ).

Chemical composition of the essential oil
The essential oil of mature Tunisian N. sativa seeds obtained using hydrodistillation was isolated in high yield (0.5%). Results of GC and GC-MS analysis of the essential oil (Table 1) indicate that the essential oil was characterized mainly by monoterpenes, with a relative total concentration of 86.5%. Sesquiterpenes represented only 0.9% of the oil. The major constituent of the oil was the hydrocarbon monoterpene p-cymene, with a relative concentration of 60.5%. The Tunisian population sampled therefore displayed a different volatile profile when compared with that of N. sativa from Iran, Morocco and Algeria (Nickavar et al., 2003;Moretti et al., 2004;Benkaci-Ali et al., 2007). It has been reported that the chemical compositions of the essential oil are highly influenced by climatic conditions and geographical factors (Sangwan et al., 2002;Burt, 2004). The high level of p-cymene in the essential oil could contribute to the valorization of Tunisian N. sativa species, since this monoterpene is of great importance in industry as intermediate for synthesis of fragrances, pharmaceuticals and herbicides (Martin-Luengo et al., 2008).

Antioxidant activity
Reactive oxygen species (ROS), including oxygen radicals and their reaction products, are known to react with biological molecules, leading to cell and tissue damage. In vitro methodologies have been widely used to assess antioxidant properties of medicinal plant extracts, however, these assays are often very specific for a particular mode of action and do not necessarily reflect the normal biological context in which they react (Girard-Lalancette et al., 2009). Recently, the cell-based assay using DCFH-DA, a useful indicator of reactive oxygen species (ROS), has been developed as a new sensitive test which allows detection of both the anti-and pro-oxidant properties (Girard-Lalancette et al., 2009). The results, presented in Table 2,  (87) demonstrated that the essential oil strongly inhibited t-BuOOH induced DCFH oxidation with an IC 50 value of 1.0 µg/ml. This result indicates that N. sativa essential oil significantly inhibits ROS production and thus exhibits the ability to protect cells from oxidative stress. The antioxidant activity of the main components present in N. sativa seed essential oil revealed that thymoquinone possesses a strong ex vivo antioxidant activity with an IC 50 value of 1.0 µM (0.2 µg/ml), while the activities of other terpenoids were weak. Surprisingly, the direct activity of carvacrol, which is a well known in vitro phenolic antioxidant compound (Ruberto and Baratta, 2000), was found to be low with an IC 50 of 190.0 µM (28.5 µg/ml). In comparison, the positive control, quercetin, exhibited an IC 50 value of 0.1 µM, which corresponds to 0.04 µg/ml. The monoterpene hydrocarbons, p-cymene, γ-terpinene and β-pinene were inactive (Table 2), despite previous reports of their in vitro antioxidant activities (Ruberto and Baratta, 2000). The antioxidant activities of these volatiles in cellular assays have not been previously reported and the results could be explained by the fact that in vitro tests do not take the physiological conditions of the cell, bioavailability of the antioxidant molecule, as well as general cellular metabolism into account (Girard-Lalancette et al., 2009). Carvacrol and thymoquinone have been reported to contribute to the in vitro antioxidant activity of N. sativa essential oil (Burits and Bucar, 2000). However, our results indicated that the ex vivo antioxidant activity of the essential oil is mainly due to the action of thymoquinone.

Anti-inflammatory activity
The anti-inflammatory activities of N. sativa seed essential oil and its major constituents were evaluated by measuring their capacity to inhibit cellular NO generation. Nitric oxide is an endogenous free radical species that is synthesized from L-arginine by nitric oxide synthase (NOS) in various tissues. This radical is an important regulator of physical homeostasis, whereas large amounts have been closely correlated with the pathophysiology of a variety of diseases and inflammations (Marletta, 1993). Therefore, the inhibition of NO production may be a useful strategy for the treatment of various inflammatory disorders (Choi et al., 2007).
The anti-inflammatory activity of N. sativa seed essential oil was evaluated on RAW 264.7 macrophages which were stimulated to induce an overproduction of NO. As presented in Fig. 1, the essential oil showed a strong inhibitory effect on LPS-induced NO secretion with 90.0% inhibition observed at 25.0 µg/ml and an IC 50 value of 6.3 µg/ml. Comparatively, the L-NAME, used as positive control inhibited NO release by 45.7% at 250.0 µM (67.4 µg/ml). The cytotoxicity of the essential oil towards RAW 264.7 cells was also evaluated. The results revealed that doses up to 25.0 µg/ml produced no significant cytotoxic effect and the cells remain viable (data not shown).
To understand the relationship between the activity of the essential oil and its composition, the main volatiles present in N. sativa seeds were tested for their anti-inflammatory properties at non-cytotoxic concentrations. Thymoquinone was found to be the most active compound, inhibiting NO production by 95.0% at 25.0 µM (4.1 µg/ml). Literature data indicate that thymoquinone mediates its inhibitory effect on NO production via the reduction of inducible NOS mRNA and protein expressions (El-Mahmoudy et al., 2002). However, ours results (Table 2; Fig. 1) suggest that the anti-inflammatory capacity of thymoquinone could be mediated, at least in part, by its strong direct antioxidant activity as an effective ROS scavenger.

Tested compounds
Inhibition of DCFH oxidation N. sativa essential oil Values are mean ± S.D. of three replications. a IC 50 values (µg/ml). b IC 50 values (µM). A relationship between the inhibition of cancer cell proliferation and inflammatory mediator production by extracts has been hypothesized. Indeed, inflammatory mediators, such as NO and cytokines, have been reported to contribute to mutagenesis (Marletta, 1993). It has been shown that the activity of the enzyme NOS is consistent in human cancer and its selective modulation has been suggested as a potential strategy for chemoprevention and reduction of cancer cell proliferation (Ahmad et al., 1997;Nishikawa et al., 2004). In this study, the effect of N. sativa seed essential oil and its major constituents were evaluated on the growth of cancer cell lines.

Anticancer activity
The anticancer activity of N. sativa essential oil was evaluated against human lung carcinoma A-549 and colon adenocarcinoma DLD-1 cell lines. Table 3 indicates that the oil was active against A-549 and DLD-1, with IC 50 values of 43.0 and 46.0 µg/ml, respectively. However, Islam et al. (2004) tested the essential oil of N. sativa from Bangladesh against other cancer cells and reported higher IC 50 values (from 120 to 384 µg/ml), which suggests that essential oil from Tunisian seeds may be more effective as anticancer agent.
The assessment of the cytotoxicity of the main compounds present in the oil showed that thymoquinone was the most active, inhibiting tumour cell lines growth with IC 50 values of 13.0 µM (2.1 µg/ml) for A-549 and 5.9 µM (1.0 µg/ml) for DLD-1. In comparison, the positive control etoposide, exhibited IC 50 values of 3.4 (2.0 µg/ml) and 27.0 µM (15.9 µg/ml) against A-549 and DLD-1, respectively (Table 3). These results confirm the cytotoxicity of thymoquinone against the A-549 cell line (Rooney and Ryan, 2005), and show for the first time its strong activity against the colon adenocarcinoma cell line, DLD-1. Results showed also that longifolene inhibits the growth of both tumour cell lines. The A-549 cells were found to be significantly more sensitive (IC 50 of 59.0 µM) in comparison with DLD-1 (IC 50 of 81.0 µM). β-pinene exhibited moderate antiproliferative activity against A-549 cell line (IC 50 of 85.0 µM) and a weak activity against DLD-1 cell line. Literature data indicated that β-pinene possesses moderate antiproliferative activity against human erythroleukemic K562 cells (Lampronti et al., 2006). However, the cytotoxicity of the sesquiterpene, longifolene, against tumour cell lines is reported for the first time.

Antibacterial activity
In this study, the antimicrobial activity of the essential oil obtained from Tunisian N. sativa seeds was investigated against S. aureus and E. coli. The results presented in Table 4 indicate that the essential oil was active against both strains with IC 50 values of 12.0 µg/ml for S. aureus and 62.0 µg/ml for E. coli.
The evaluation of activities of the volatile compounds present in the oil suggested that thymoquinone and longifolene were largely responsible for the activity of the oil against S. aureus, since they were strongly active with IC 50 values of 1.8 µM (0.3 µg/ml) and 3.0 µM (0.6 µg/ml), respectively. Thymoquinone demonstrated good activity against E. coli with an IC 50 of 41.0 µM (6.7 µg/ml). In comparison, the positive control chloramphenicol, exhibited IC 50 values of 7.0 µM (2.3 µg/ml) against S. aureus and 0.8 µM (0.3 µg/ml) against E. coli. Studies regarding the antimicrobial activity of thymoquinone are scarce; Inouye et al. (2006) studied the activity of various quinones using vapour and solution contact assays, and demonstrated that thymoquinone is a potent antifungal and antibacterial agent. To the best of our knowledge, this report is the first detailing the antimicrobial activity of longifolene.
Although thymoquinone and longifolene were highly active, other monoterpenes present in N. sativa seed essential oil, such as the major constituent p-cymene, might be critical to the activity. Despite the finding that p-cymene was only weakly antibacterial in our study, it has been demonstrated to work synergistically with carvacrol and a mixture exhibited greater antibacterial activity than the terpenoids on their own (Ultee et al., 2000).
Antibacterial and cytotoxic properties of N. sativa essential oil could be due to the ability of the oil to permeabilize membranes, including mitochondrial membranes, and to destroy cellular integrity of bacteria and eukaryotic cells, leading to cell death by necrosis and apoptosis (Bakkali et al., 2007). Table 3 Cytotoxic activities of Nigella sativa seed essential oil and its main constituents against A-549 and DLD-1 cell lines.
Tested compounds S. aureus E. coli N. sativa essential oil 12.0 ± 4.0 a 62.0 ± 17.0 a p-Cymene In conclusion, these data indicate that the essential oil extracted from Tunisian black cumin seeds exhibit potent biological activities, which support their use in traditional medicine. In addition, the essential oil might be useful for therapeutic purposes to prevent ROS disorders, to treat chronic inflammatory pathologies associated with overproduction of nitric oxide and for use as an anticancer and antibacterial agent. Moreover, results regarding the bioactivities of the main volatile components suggest that the observed activities of the essential oil are connected to its chemical composition, where thymoquinone and longifolene has been found to be the most active compounds. Furthermore, this study proved for the first time, the potential of some terpenes as direct antioxidant (thymoquinone), anti-inflammatory (carvacrol, p-cymene, β-pinene, γ-terpinene and longifolene), anticancer and antibacterial (longifolene) principles with a potential use in pharmaceuticals.