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

Abstract The essential oil of Ducrosia ismaelis Asch. (Apiaceae) that grows wild in Saudi Arabia was investigated utilizing gas chromatography (GC), and gas chromatography-mass spectrometry. Fifty constituents were characterized, representing 96.1% of the total oil. The D. ismaelis essential oil (DIEO) was distinguished by a high composition of oxygenated monoterpenes (51.6%). Decanal (40.6%), α-pinene (15.1%) and dodecanal (13.7%) were the fundamental components. Additionally, DIEO was evaluated for its cytotoxic, antibacterial, antifungal and antioxidant activities. DIEO revealed a great cytotoxic effectiveness against the tested cancer cell lines with IC50 values between 66.2 and 137.3 μg/mL particularly against MCF-7 cancer cells. Furthermore, the induction of apoptosis against MCF-7 cells has been asserted using staining assay (annexin VFITC and/or propidium iodide (PI) dyes) and flow cytometry technique. The DIEO possessed a strong antimicrobial activity against Gram-positive bacterial and fungal strains with MIC-values between 0.07 and 0.31 mg/ml. The values of MBC or MFC were almost once higher than those of MIC’s. Moreover, the β-carotene-bleaching and DPPH free radical-scavenging tests showed that DIEO had a moderate activity (68%) as an antioxidant agent in decolouring of the β-carotene at 1.0 mg/mL and a moderate radical scavenging for DPPH (66 and 72%) at 0.50 and 1.0 mg/mL.


Introduction
The genus Ducrosia belongs to the family Apiaceae and comprises only six species: D. anethifolia (DC.) Boiss., D. flabellifolia Boiss., D. ismaelis Asch., D. assadii Alava., D. areysiana Pimenov & Kljuykov, and D. inaccessa Pimenov and Kljuykov, which grow wild in Africa and Asia particularly in Iran, Iraq, Syria, Pakistan, Afghanistan and countries along the Arabian Gulf [1][2][3]. The genus of Ducrosia is represented in Saudi Arabia by three species namely D. anethifolia, D. flabellifolia and D. ismaelis. D. ismaelis is indigenous to the central province of Saudi Arabia [4,5]. This species is a small, glabrous, perennial plant with herbaceous branches from a woody base [5].
The aerial parts of this plant have a distinctive aroma and have been applied in the traditional folk medicine in Saudi Arabia to treat skin infections and as a repellent agent for insects and reptilians [5]. Generally, different species of Ducrosia are utilized as pain killers and in common cold treatments in traditional medicine [6]. Moreover, various pharmacological activities e.g. antioxidant, antimycobacterial, antianxiety, central nervous system depressant, antibacterial and antifungal have been accounted for several Ducrosia species, including D. anethifolia, D. assadii, D. flabellifolia and D. ismaelis [7][8][9][10].
In spite of the utilization of D. ismaelis conventionally in herbal medicine in Saudi Arabia, there is a lack of knowledge on the pharmacological activities and biochemical compounds of the essential oil of D. ismaelis.
Analysis of previous studies available shows that only a single scientific study has been done regarding the chemical compositions of the essential oil of D. ismaelis growing in Saudi Arabia. Continuing our investigations of aromatic plants that widely grow in our region, we illustrate here the cytotoxic, antibacterial, antifungal and antioxidant potentials, and chemical content of the essential oil of D. ismaelis (DIEO).

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.

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.

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.

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.

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).

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% CO 2 . Shortly, one mL of cell suspension (5 X 10 4 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 IC 50 of DIEO was measured from the dose-response curves using the following formula: % Cell Viability= Mean absorbance of DIEO / Mean absorbance of control x 100

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 IC 50 concentration. The observation of the morphological alterations of the apoptotic cells were achieved utilizing phase contrast inverted microscope (Evos, USA).

Apoptosis assessment by DAPI staining and acridine orange/ethidium bromide assays
MCF-7 cells were handled with IC 50 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.

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 IC 50 . 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.

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.  [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.

β-Carotene bleaching assay
The potential of DIEO as antioxidant agent has been investigated with the β-carotene bleaching test as reported previously by Mothana and co-workers [25]. The prepared solution of β-carotene (1 mL of 200 μg dissolved in 1 mL chloroform) was added to a solution of 200 μL of Tween-20 and 20 μL of linoleic acid in a flask, followed by evaporation of the chloroform. Then, distilled water (100 mL) was added into the flask and mixed for 2 min. 200 μL of the DIEO (1000 μg/mL) was mixed with 2 mL of the β-carotene-linoleic acid emulsion and incubated at 40°C for 2 hours. Finally, the absorbance was read at intervals of 30 min at 470 nm. Rutin (1000 μg/mL) was the positive control. The following formula determined the antioxidant activity: Antioxidant activity (%) = 1-(Abs0 -Abst) / (Abs°0 -Abs°t) X 100 where, Abs0 and Abs°0 are the optical densities of DIEO and blank samples respectively, that recorded at zero time of incubation. Abst and Abs°t are the optical densities determined for DIEO and blank samples at 120 min, respectively.

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.

Results
The aerial part of D. ismaelis afforded a light yellow oil (0.28% w/w) with a strong aromatic odour.

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).

Cytotoxicity and apoptosis staining
The essential oil of D. ismaelis (DIEO) exhibited a noteworthy cytotoxic potential against the tested cancer cell lines with IC 50 values ranging between 66.24 to 137.32 μg/mL (Table 2   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 IC 50 . 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.

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 MICvalues 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.

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).

Discussion
Volatile oils are commonly used in the traditional medicine throughout human history. The use of this type of natural products include a wide range of curative and hygienic applications. Several investigations in various cell and animal models have shown the efficacy of the volatile oils as antimicrobials, antioxidants, anti-inflammatory and anti-cancer agents [26]. Investigations on aromatic medicinal plants therefore need to be pursued in order to explore novel and more effective remedies. Consequently, this study has been conducted in our search for substantial and hopeful natural products from medicinal plants grown in the Middle East. In the current work, we evaluated the chemical content of the essential oil of D. ismaelis by GC/ FID and GC/MS. Furthermore, the existing investigation has been expanded to assess the cytotoxic, antimicrobial and antioxidant potentials and to report the conceivable apoptotic passageway with Annexin VFITC and/or propidium iodide (PI) dyes and flow cytometer analyses. The obtainable information on the species D. ismaelis is generally limited. Only one single report was found   on the chemical composition of the essential oil of D.
In the existing investigation, we observed auspicious cytotoxic, antimicrobial and antioxidative activities for DIEO. DIEO's cytotoxic effect was evaluated against three different cancer cell lines (MCF-7, HepG2 and LoVo) utilizing MTT assay. DIEO showed remarkable cytotoxic activity against the three cancers investigated, in particular MCF-7 cancer cells. Overall, data on cytotoxicity of DIEO are still rare however, there are few reports on the cytotoxicity of other Ducrosia species e.g. D. flabellifolia, D. anethifolia which indicated good to moderate cytotoxic activity of the isolated essential oils [14]. Additionally, Talib and co-workers [7] reported a promising activity against MCF-7 cell line with IC 50 value of 25.3 μg/mL for the ethanolic extract of D. flabellifolia [7]. They also observed that the anticancer impact was exerted by inducing programmed cell death (apoptosis) as demonstrated by the fragmentation of DNA and apoptotic bodies formation in treated cancer cells [7].
Apoptosis (programmed cell death) is a physiological process in which harmed or abnormal cells can be discarded. Consequently, the induction of apoptosis appears to be an appealing aim to kill tumour cells and is recognized, therefore, as a useful strategy in both treatment as well as prevention of cancers [30][31][32][33]. In the existing study, the morphological alterations of apoptotic cells have been monitored utilizing fluorescence microscopy after double staining with acridine orange and ethidium bromide (AO/ EtBr) and DAPI fluorescence. Furthermore, the apoptosis was proven utilizing flow cytometry after staining with annexin V-FITC/PI. While untreated cells, on the contrary, exhibited normal morphology DIEO-treated celled showed morphological changes. The main characteristics observed in our investigation, involve cell shrinkage, chromatin condensation and DNA fragmentation. An earlier study [34] carried out by Liu and co-workers (2012) indicated a strong cytotoxic effect against Hela cell line for the sweet orange oil and the isolated decanal with IC 50 values of 5.5 and 4.5 μg/mL, respectively. Moreover, compounds such as carvacrol, α-pinene, thymol and eugenol showed a cytotoxic activity against several cancer cells e.g. HepG2, MCF-7 and MDA-MB-468 [35][36][37]. Accordingly, the observed cytotoxic activity of DIEO could probably be attributed to decanal and α-pinene which were the fundamental constituents of DIEO. In our experiments, observed antimicrobial activity for DIEO was also in agreement with the results by Al-meshal who reported antimicrobial activity for the essential oil of D. ismaelis [6]. Liu and co-workers [34] reported interesting antimicrobial and antioxidant activities for decanal isolated from sweet orange oil. The liposolubility of DIEO and the major compounds may affect membrane fluidity and cause degradation or destruction of cell membrane, and this in turn, can lead to antimicrobial and cytotoxic activity [34,38].

Conclusion
The analyses with GC and GC/MS concluded that the chemical composition of D. ismaelis essential oil (DIEO) was high in oxygenated monoterpenoids where the fundamental constituents were decanal (40.6%), α-pinene (15.1%) and dodecanal (13.7%). The results obviously revealed that DIEO has interesting cytotoxic, antimicrobial and mild antioxidant activities. The acquired results proposed that DIEO induced apoptosis in MCF-7 cells.
Our findings, therefore, support the assumption that DIEO may be a hopeful cytotoxic and antimicrobial agent. Additional experiments are required to isolate the active principles and to reveal the mechanism of actions.