Ducrosia ismaelis Asch. essential oil: chemical composition profile and anticancer, antimicrobial and antioxidant potential assessment

2.1 Plant material

The aerial part of D. ismaelis was collected from the Riyadh region in Saudi Arabia in January 2016. The authentication of D. ismaelis was performed at the department of Pharmacognosy, College of Pharmacy, King Saud University (KSU) and a voucher specimen (KSU 16240) was stored in the herbarium of the college.

2.2 Isolation of the volatile oil

The dried aerial part of D. ismaelis (300 g) was coarsely ground, placed in a round flask containing 500 ml of water, and distilled utilizing a Clevenger apparatus for 4 h. A desiccation of the acquired essential oil was done over anhydrous sodium sulphate. After filtration, the oil was stored at +4°C until investigation.

2.3 Gas Chromatography/Mass spectrometry investigation

Gas chromatographic analysis was conducted with a Gas Chromatograph (GC 5975) connected with a mass spectrometer (Agilent, USA; SEM Ltd., Istanbul, Turkey). Innowax FSC column (60 m x 0.25 mm, 0.25 μm film thickness) was utilized as stationary phase. The mobile phase in this experiment was helium with a flow rate of 0.8 mL/min. The injection volume was 0.1 µL with a split ratio of 40:1. The temperature of the oven was set at 60°C for 10 min, thereafter raised constantly (4°C /min) to reach 220°C, maintained constant for 10 min and then raised slowly (1°C /min) to reach 240°C. The detection of mass spectra was done at 70 eV with scan mass extent of m/z 35-450.

2.4 Gas Chromatography/FID investigation

The analysis was conducted with the gas chromatograph system with FID detector (an Agilent Technologies 6890 N GC). The same stationary and mobile phases used in GC–MS experiment and same operating conditions were executed in triplicate. The FID temperature was adjusted to 300°C. Auto injection was implemented simultaneously to acquire equivalent retention times. Quantitative data was calculated from the FID peak area percent normalization.

2.5 Chemical content

The identification of the chemical compounds of the DIEO was achieved by the comparison of the MS with those of similar components based on their indexes of retention using Adams library [18], Mass Finder terpenoid library [19], Wiley GC/MS Library [20] and our own Baser Library of Volatile Oil Constituents. The DIEO compounds were specified by comparison of the retention times with genuine reference compounds and by comparing the retention index (RRI) relative to C8-C30 of n-alkanes under the operational conditions described above [21]. The results obtained from three replicates were statistically analysed and arranged in Table 1 (mean ± standard deviation).

2.6 Determination of cytotoxic potential (MTT assay)

The cytotoxic potential of DIEO was investigated utilizing MTT assay [22] against three diverse human cancer cell lines included HepG2 (liver-cancer), LoVo (colon-cancer) and MCF-7(breast-cancer). The cells were incubated using DMEM enhanced with 10% FBS at 37°C with 5% CO₂. Shortly, one mL of cell suspension (5 X 10⁴ cells/mL) was seeded in 24-well plate. The treatment with different concentrations of DIEO was performed after the cultivation of the cells for 24 hours. Then, each well of the plates received 100 µL of MTT solution (5 mg/ml) and incubated at 37°C for 2-4 hours. The measurement of the diminished MTT was achieved at 540 nm with microplate ELISA reader (Thermo Fisher Scientific, USA); wells containing cells treated with DMSO (1%) were considered as controls. Vinblastine was utilized as a positive control. The IC₅₀ of DIEO was measured from the dose-response curves using the following formula:

2.7 Light microscopy study

MCF-7 cells have been cultivated using 12-well plates, followed by the incubation for 24 h and the treatment with and without DIEO at the IC₅₀ concentration. The observation of the morphological alterations of the apoptotic cells were achieved utilizing phase contrast inverted microscope (Evos, USA).

2.8 Apoptosis assessment by DAPI staining and acridine orange/ethidium bromide assays

MCF-7 cells were handled with IC₅₀ of DIEO for 24 h, then washed with phosphate buffered saline (PBS) and fixed with ethanol for 15 min at 25°C. After that cells were stained with DAPI (2 μg/mL) and incubated again in the dark for 30 min. Finally, MCF-7 cells were investigated and imaged utilizing a fluorescence microscope (Evos, USA). In order to perform the acridine orange/ethidium bromide staining test, 2 µL of acridine orange/ethidium bromide (one part each of 3 mg/mL acridine orange and 3 mg/mL ethidium bromide in PBS) was mixed with 1 mL suspension of MCF-7 cell in a 12-well plate. The examination of the cells was achieved on an EVOS® imaging connected to a digital imaging system.

2.9 Flow cytometry analysis of cell apoptosis

The apoptosis was detected using the procedure depicted in the manufacturer’s instructions of Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit (Thermo Fisher Scientific, Inc.). In short, MCF-7 cells were seeded for adherence in a tissue culture plate (6-well) at a density of 1x106 cells/well at 37°C for 24 hours, and then were treated with DIEO IC₅₀. After a 24 h treatment with DIEO, the cells were gathered, washed twice in cold PBS and resuspended in kit specific binding buffer. Then, the cells were incubated in the dark with 5 μL of Annexin V-FITC and 5 µL of PI for 15 min. Finally, 400 µL of annexin-binding buffer was given to the cells and promptly analysed using flow cytometry.

2.10 Determination of antimicrobial activity (Determination of MIC, MBC, MFC)

The effectiveness of DIEO as an antimicrobial agent was determined using two-fold micro-dilution assay following by cultivation method for measuring MIC, MBC and MFC. The microorganisms used in the present work included Staphylococcus aureus TCC 25923, Staphylococcus epidermidis ATCC 12228)), Escherichia coli ATCC 25922, Acintobacter sp. ATCC 49139, Aspergillus ochraceus AUMC 9478, Penicillium chrysogenum AUMC 9476, Candida albicans ATCC 60193 and Rhodotorula sp. Wild type [23]. The MIC (minimal inhibition concentration) of DIEO was assigned as the lowest concentration exhibiting no tangible microbial growth. Gentamycin and nystatin (125 to 0.97 µg/mL) were applied as positive antibacterial and antifungal controls. The minimal bactericidal concentration (MBC) and minimal fungicidal concentration (MFC) were assessed among the concentrations that had ability to kill all microbial cells in wells using the sub-cultivation method.

2.11 Determination of antioxidant activity

2.12 Experimental design and statistical analysis

The present experiment was carried out in a completely randomized design (CRD) and the mean and standard deviations (SD) (N=3) were calculated. The significant differences were evaluated using the Tukey test in one-way ANOVA at P < 0.05 (IBM, SPSS, statistics 25).

Ethical approval: The conducted research is not related to either human or animal use.

3.1 Chemical content of DIEO

The results of the GC/FID and GC/MS analysis are shown in Table 1. The components of the oil, their retention indices, percentage composition and identification methods are summarized in Table 1. The GC/FID analyses of the D. ismaelis essential oil (DIEO) led to the identification of fifty compounds, representing 96.1% of the total oil. The oil (DIEO) was characterized by a high content of oxygenated monoterpenes (51.6%) followed by monoterpene hydrocarbons (23.4%). Decanal (40.6%), α-pinene (15.1%) and dodecanal (13.7%) were the major constituents found in DIEO (Table 1, Figure 1).

Figure 1

Major constituents of DIEO.

3.2 Cytotoxicity and apoptosis staining

The essential oil of D. ismaelis (DIEO) exhibited a noteworthy cytotoxic potential against the tested cancer cell lines with IC₅₀ values ranging between 66.24 to 137.32 μg/mL (Table 2 and Figure 2). The greatest activity was demonstrated against MCF-7 cancer cells with IC₅₀ value of 66.24 μg/mL. All tested cells showed a dose-dependent decrease in cell proliferation (Figure 2). The morphology of MCF7 cells was examined after DIEO treatment to determine if any changes occurred in the appearance of cell morphology. The treatment of MCF7 cells with the IC₅₀ concentration of the DIEO for 24 h had significant effect on the appearance and morphology of the MCF-7 cells including decreased densities of the cells, shrinkage detachment and loss of cell integrity compared to cells cultiviated with DMSO vehicle control (Figure 3). Furthermore, there was a significant decrease in cell number of the treated cells. Two cell staining assays (DAPI and acridine orange/ethidium bromide) were employed to examine the effect of DIEO on the nuclear morphological changes in treated MCF7 cells after treatment with IC₅₀. MCF-7 cells treated with a DMSO-vehicle control showed primarily nuclear staining with acridine orange and no ethidium bromide staining, while treatment with the DIEO for 24 h exhibited a change in cell morphology including changes in nuclear staining corresponding to condensation and presence of some staining with ethidium bromide (Figure 3). The representative signs of apoptosis in the treated cells were also obvious (Figure 3). The induction of apoptosis was affirmed quantitatively utilizing flow cytometer analysis. The labelling of the cells was achieved with Annexin VFITC and/or propidium iodide (PI) dyes (Figure 4). DIEO mediated apoptosis in treated MCF-7 cell lines at 24 hours of handling. There was no incidence of apoptosis (0 percent) in untreated cells suggesting that the DIEO is the source of apoptosis induction (Figure 4). As the treatment time increased to 24 hours, the apoptotic cells percentage (early and late) was also raised in proportion to the exposure time.

Figure 2

Effect of DIEO on growth inhibition of MCF7, HepG2 and LoVo cell lines. The cells were incubated with increasing concentrations of DIEO, the cell growth was measured using the MTT assay after 48h. The data shows mean values ± standard deviation (SD) of at least 3 independent experiments. The data were analyzed using Origin 8 Pro and significant differences confirmed by student T-test * (P < 0.05).

Figure 3

Morphological changes and apoptosis detection in MCF 7 cells treated with DIEO. MCF7 cells were cultured and treated with the IC₅₀ of DIEO for 24h. (A) Morphological changes seen under light phase contrast microscope, (B) Apoptotic cells were investigated by fluorescence DAPI staining, (C) Cells were stained with a mixture of (AO: Acridine Orange, EtBr: Ethidium Bromide). Viable cells excluded EtBr and their nuclei were bright green, while apoptotic cells were orange to red color.

Figure 4

Apoptosis detection using flow cytometry after annexin V-FITC/propidium iodide (PI) staining for (a) Control and (b) DIEO. Viable cells are in the lower left quadrant (AA3); early apoptotic cells are in the lower right quadrant (AA4); late apoptotic cells are in the upper right quadrant (AA2); and non-viable necrotic cells are in the upper left quadrant (AA1).

3.3 Antimicrobial activity

The antimicrobial potential against bacterial and fungal strains is shown in Table 3. Minimal inhibitory concentrations (MICs), minimal bactericidal concentration (MBCs) and minimal fungicidal concentration (MFCs) of DIEO are shown in Table 3. The DIEO exhibited different growth inhibitions of the microbial strains with MIC-values ranging from 0.07 to 5.0 mg/mL. The values of MBC or MFC were, once, almost higher than those of MIC’s (Table 3). While the Gram-negative bacterial strains e.g. E. coli showed less sensitivity with MIC-value of 2.5 mg/mL, the most prone microbial strain (MIC: 0.07 mg/mL) was the Gram-positive S. aureus followed by the fungal strains (0.15 to 0.31 mg/mL). The Gram-negative bacterial strains e.g. E. coli showed less sensitivity with MIC-value of 2.5 mg/mL.

3.4 Antioxidant activity

The results of the radical scavenging and antioxidative potential of DIEO are displayed in Table 4. DIEO exhibited a marked power in the β-carotene-bleaching assay to diminish the β-carotene bleaching at of 1.0 mg/mL with a total antioxidant value of 68% (Table 4). In addition, DIEO had a notable free radical scavenging (66 and 72%) at the concentrations 0.5 and 1.0 mg/mL (Table 4).