Cytotoxicity of the Essential Oil of Fennel (Foeniculum vulgare) from Tajikistan

The essential oil of fennel (Foeniculum vulgare) is rich in lipophilic secondary metabolites, which can easily cross cell membranes by free diffusion. Several constituents of the oil carry reactive carbonyl groups in their ring structures. Carbonyl groups can react with amino groups of amino acid residues in proteins or in nucleotides of DNA to form Schiff’s bases. Fennel essential oil is rich in anise aldehyde, which should interfere with molecular targets in cells. The aim of the present study was to investigate the chemical composition of the essential oil of fennel growing in Tajikistan. Gas chromatographic-mass spectrometric analysis revealed that the main components of F. vulgare oil were trans-anethole (36.8%); α-ethyl-p-methoxy-benzyl alcohol (9.1%); p-anisaldehyde (7.7%); carvone (4.9%); 1-phenyl-penta-2,4-diyne (4.8%) and fenchyl butanoate (4.2%). The oil exhibited moderate antioxidant activities. The potential cytotoxic activity was studied against HeLa (human cervical cancer), Caco-2 (human colorectal adenocarcinoma), MCF-7 (human breast adenocarcinoma), CCRF-CEM (human T lymphoblast leukaemia) and CEM/ADR5000 (adriamycin resistant leukaemia) cancer cell lines; IC50 values were between 30–210 mg L−1 and thus exhibited low cytotoxicity as compared to cytotoxic reference compounds.


Introduction
Fennel (Arpabodiyon, local Tajik name), Foeniculum vulgare Miller, an important member of the Apiaceae, is widely used for flavouring foods and beverages due to its pleasant spicy aroma [1,2]. In traditional medicine, the plant and its essential oil have been extensively used as carminative, digestive, galactogogue and diuretic and to treat respiratory and gastrointestinal disorders [1]. It is also used as a constituent in cosmetic and pharmaceutical products [3]. The essential oil of F. vulgare, in particular anethole, exhibits antispasmodic, carminative, anti-inflammatory, estrogenic and anti-microbial activities [4]. In vitro, fennel oil possesses antioxidant [5,6], antimicrobial [7], insecticidal [8], antithrombotic [9] and hepatoprotective activities [2]. Furthermore, the essential oil of fennel exhibits in vitro anticancer activity [10][11][12]. The in vitro cytotoxic, genotoxic, and apoptotic activities of estragole were suspected to induce hepatic tumors in susceptible strains of mice [10].
Anethole is toxic in high concentrations [4]. Because of their lipophilic properties, the secondary metabolites of essential oils are able to penetrate cytoplasmic membranes by free diffusion. This process can affect membrane fluidity and permeability, transport, ion equilibrium and membrane potential [13], leading to cell death by apoptosis and necrosis [11].
The essential oil of fennel is rich in secondary metabolites, which carry reactive substituents (among them carbonyl groups) in their ring structures or side chains. Aldehydes are generally long-lived and electrophilic compounds, they can react with molecular targets which carry free amino groups, such as of amino acid residues in proteins or of nucleotides in DNA to form Schiff's bases [14]. Aldehyde-containing essential oils often exhibit cytotoxicity [15,16] by reacting with cellular nucleophiles, including proteins and nucleic acids [13,17].
The chemical composition of the essential oil of F. vulgare from different geographical locations has been extensively studied [6,[18][19][20]. According to these studies, the major components of fennel oil are trans-anethole, estragole, fenchone, and limonene depending on the chemotype [21][22][23]. The aim of the present study was to investigate the chemical composition of the essential oil of fennel growing in Tajikistan (Central Asia) and to explore cytotoxic activity against different human cancer cell lines. The biological activity and chemical composition of F. vulgare oil from Tajikistan have not been previously reported.

Plant Material
The aerial parts of F. vulgare plants were collected from the Varzob region, Tajikistan on 29 July 2016. A voucher specimen of the plant material was deposited at the Department of Pharmaceutical Technology, Avicenna Tajik State Medical University under accession number TD2016-24. The material was completely dried and hydrodistilled using a Clevenger-type apparatus for 3 h to give an essential oil yield of 0.5%.

Gas-Liquid Chromatography-Mass Spectrometry (GLC-MS)
The essential oil from F. vulgare oil was analyzed by GLC-MS using an instrument (GCMS-QP2010 Ultra, Shimadzu, Tokyo, Japan) operated in the EI mode (electron energy = 70 eV), scan range = 3.0 scans s −1 . The GC column was ZB-5 fused silica capillary with a (5% phenyl)-polymethyl siloxane stationary phase a film thickness of 0.25 mm. The carrier gas was helium with a column head pressure 551 kPa and flow rate of 1.37 mL min −1 . Injector temperature was 250 • C and the ion source temperature was 200 • C, increased in temperature rate 2 • C min −1 to 260 • C. The GC oven temperature program was programmed from 50 • C initial temperature, increased at a rate of 2 • C min −1 to 260 • C. A 5% w/v solution of the sample in CH 2 Cl 2 was prepared and 0.1 µL was injected in splitting mode (30:1).
Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes (Kovats RI), and by comparison of their mass spectral fragmentation patterns with those reported in the literature [24] and stored on the MS library (NIST 11 (National Institute of Standards and Technology, Gaithersburg, MD, USA), WILEY 10 (John Wiley & Sons, Inc., Hoboken, NJ, USA), FFNSC version 1.2 (Shimadzu Corp., Tokyo, Japan)). The percentages of each component are reported as raw percentages based on total ion current without standardization (set 100%).

Cytotoxicity
The potential cytotoxicity of the fennel essential oil against of the five human tumor cell lines (HeLa, Caco-2, MCF-7, CCRF-CEM and CEM/ADR5000) were determined by the MTT assay. The cells were seeded at a density of 2 × 10 4 cells/well (HeLa, Caco-2, MCF-7) and 3 × 10 4 cells/well (CCRF-CEM and CEM/ADR5000). The essential oil was serially diluted in media in the presence of DMSO at concentrations between 10 mg/L and 5 g/L; 100 µL of each concentration was applied to the wells of a 96-well plates. Cells were incubated with the essential oil for 24 h (HeLa, Caco-2, MCF-7) and 48 h (CCRF-CEM and CEM/ADR5000) before the medium was removed and replaced with fresh medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The formazan crystals produced were dissolved in DMSO 4 h later; the absorbance was measured at 570 nm with a Biochrom Asys UVM 340 Microplate Reader (Cambridge, UK).

Hemolytic Activity
The hemolytic activity was investigated by incubation of serially diluted fennel essential oil in phosphate-buffered saline with red blood cells (human O+). The hemolytic activity assay was performed as described earlier [27].

Microscopic Observation
The images of the treated or untreated CCRF cells were obtained and photographed using a by fluorescence microscopy (BZ-9000, Keyence, Osaka, Japan) in order to investigate morphological changes.

Data Analysis
The experiments were repeated three times. IC 50 values were calculated using a four parameter logistic curve (Sigma Plot 11.0 (SYSTAT Software, San Jose, CA, USA)). The data are represented as means ± standard deviations. The results of statistical test were determined by using Sigma Plot 11.0 software and also by using the statistical function t-test in Microsoft Excel. A p value below 0.05 was considered to represent statistical significance.

Chemical Composition
The essential oil of F. vulgare was analyzed by gas-liquid chromatography-mass spectrometry (GLC-MS). Thirty components were identified representing 97.7% of total oil composition (Table 1). Oxygenated terpenoids were the dominant compounds of the essential oil of F. vulgare.

Antioxidant Activity
The investigation of antioxidant activity of essential oils as lipophilic secondary metabolites became an interesting aspect of food and pharmaceutical research. Synthetic food additives are increasingly replaced with plant-based natural ingredients, due to their safety, effectiveness and consumer acceptance [50]. In general, fennel as an edible and medicinal plant represents an interest through the neutralization of reactive oxygen species in order to prevent the damage of protein, lipid, and DNA which are supposed to be the main reason for cell aging, oxidative stress-originated diseases (cardiovascular and neurodegenerative diseases), and cancer.
The essential oil of fennel exhibits low antioxidant activity as compared to the positive control, caffeic acid. The results of the DPPH, ABTS and FRAP analyses are represented Table 2. Table 2. Antioxidant activity of the essential oil Foeniculum vulgare as determined by the ABTS, DPPH, and FRAP assays *.  The concentration of 50% inhibition (IC 50 ) was the parameter used to compare the DPPH and ABTS radical scavenging activity. A lower IC 50 (for DPPH and ABTS) and higher FRAP values indicate higher antioxidant activity. IC 50 values for the antioxidant activity were 15.6 mg mL −1 (DPPH) and 10.9 mg mL −1 (ABTS). The IC 50 values of the known antioxidant substance-caffeic acid-were 0.0017 mg mL −1 for DPPH and 0.0011 mg mL −1 for ABTS, respectively. Ferric reducing antioxidant power (FRAP) were equivalent to 193.5 µM Fe(II)/mg for oil and 2380 µM Fe(II)/mg for caffeic acid. In agreement with our results, it was reported that the an IC 50 value of DPPH radical scavenging activity of Foeniculum vulgare essential oil was 15.3 mg mL −1 [7]. According to the authors [7], fennel essential oil reacts with free radicals as a primary antioxidant and, therefore, it may limit free-radical damage occurring in the human body. In our previous paper, we reported the antioxidant activity of pure essential oil components, including the main component of the essential oil of fennel (trans-anethole). It shows weak antioxidant activity. We assume that the phenolic substances (carvacrol (2.1%) and thymol (1.0%)) are responsible for the observed antioxidant activity. These data are in agreement with previously reported data [25]. However, it is known that the bioactivity of plant extracts is due to the entire composition of the extract [51,52].

Cytotoxicity
The cytotoxicity of the oil was tested against HeLa, Caco-2, MCF-7, CCRF-CEM and CEM/ADR5000 cancer cell lines (Table 3). IC 50 values were 207 mg L −1 for HeLa, 75 mg L −1 for Caco-2, 59 mg L −1 for MCF-7, 32 mg L −1 for CCRF-CEM, and 165 mg L −1 for CEM/ADR5000 cell lines. As compared to the positive control doxorubicin, the essential oil exhibits low cytotoxicity. Doxorubicin, an anthracycline antitumor antibiotic is a hydrophilic drug, and shows broad spectrum anticancer activity [53]. The cytotoxicity of F. vulgare oil is most likely due to the lipophilic properties of essential oil and alkylating properties of the major components trans-anethole and p-anisaldehyde. Caco-2 and CEM/ADR5000 overexpress the ABC transporter p-gp which can actively pump out any lipophilic compound that has entered the cell by free diffusion [54]). Thus, both cell lines are rather insensitive towards lipophilic cytotoxic agents. In contrast, the parent cell line CCRF-CEM should be sensitive. We also suspect that some components of the essential oil are may be substrates for p-gp, as IC 50 values were higher in CEM/ADR5000 cells. To better understand the mechanism of action of F. vulgare essential oil, we have investigated its hemolytic effect. The result of hemolytic activity indicates that the oil is able to lyse the cell membrane albeit with a rather high IC 50 value of 1100 mg L −1 (Table 3). Moreover, in order to investigate the effect of essential oil on the cell morphology, the images of untreated and treated CCRF cells with essential oil were captured by fluorescence microscope. The images are illustrated in Figure 2.
Obtained images indicate that the essential oil can change the morphology of cells. Results of both hemolysis and microscopic investigation indicate that essential oil also affects the integrity of cell membranes. This is in agreement with many of the reported data [55].
In addition, trans-anethole, the main component of the essential oil, was examined for its cytotoxicity in RC-37 cells. Its IC 50 value was 100 mg L −1 [56]. The incubation of hepatocytes with anethole caused a cell death accompanied by losses of cellular ATP and adenine nucleotide pools [57]. Anethole shows apoptotic activity, as it can damage DNA [58]. Thus anethole could be responsible for the overall cytotoxicity of the essential oil in our study.

Conclusions
The essential oil of fennel contains several bioreactive secondary metabolites, such as aldehydes. The oil apparently affects the stability of biomembranes and interacts with molecular targets, such as proteins and DNA, which causes a low cytotoxicity.