CHEMICAL COMPOSITION, ANTIMICROBIAL, ANTIOXIDANT AND ANTICANCER ACTIVITIES OF ESSENTIAL OIL FROM AMMODAUCUS LEUCOTRICHUS COSSON & DURIEU (APIACEAE) GROWING IN SOUTH ALGERIA

The chemical composition of the essential oil obtained by hydrodistillation from aerial parts of A. leucotrichus Cosson & Durieu (Apiaceae) grown in the south of Algeria (El-Oued) was determined by GC-MS analysis. The oil was found to be rich in perilladehyde 64.66% and D-Limonene 26.99%. The biological activity of A. leucotrichus Cosson & Durieu essential oil has been investigated. The in vitro antimicrobial activity of the essential oil sample was tested on eight strains, one yeast and one fungi. The test showed interesting antimicrobial properties, especially on Salmonella enterica and E. coli, the antioxidant capacity of the oil was measured using the cyclic voltammetry, and the AAT value of A. leucotrichus essential oil was evaluated 47.84 mg α-TE/L. In addition, the antitumor activity showed that the oil of A. leucotrichus was very significant against the HCT116 colon cancer cell line.


INTRODUCTION
Ammodaucus leucotrichus Cosson & Durieu belonging to Apiaceae family, is one of the huge plant's diversity of the Sahara desert [1]. It is a small annual plant, 10-12 cm in height, glabrous with erect, finely striates stems [2]. The leaves are finely dissected and slightly fleshy. The flowers are grouped in umbels of 2 to 4 bran. The flowers are small, with five free petals. The fruit is a diachene, 6-10 mm, long and is covered with dense silky white hairs. It usually flowers in early spring (February to April). The plant is common in the Algerian Sahara (El-Oued) [3]. In the Algerian traditional medicine, A. leucotrichus is used for the treatment of common cold and fever [4], stomach diseases, vomiting pains and allergies [5]. Moreover, emmenagogue, abortive and aphrodisiac properties are attributed to this species. The flowers are used for the treatment of cardiac diseases [6]. A. leucotrichus seeds decoction plays an important role in traditional medicine in North African countries (Algeria, Morocco, Tunisia, Libya, Egypt, Mauritania and upper Niger valley). Its best implantation is in desert regions, often down a hill or a dune [7]. The leaves are used to aromatize tea. Powdered, it is much-appreciated as a spice food in the El-Oued area [8]. Even though, this plant is known to grow wild in Algeria and in spite of its wide uses in traditional medicine, a full investigation of the chemical composition of the essential oil extracted from the aerial parts, has never been mentioned before. This study is considered as the first original report giving full details of the composition as well as the antimicrobial, anticancer and antioxidant properties using spectrophotometrical (DPPH) and electrochemical methods (cyclic voltammetry) of the aerial parts oil obtained from Ammodaucus leucotrichus Coss & Dur, to effectively exploit of the usage of this plant. The essential oil was also evaluated for its anticancer activity against HePG2 (Hepatic) and HCT116 (Colon) human cancerous cell lines.

Plant material
The plant material of Ammodaucus leucotrichus was obtained from the valley state of southeast Algeria, in February 2018. Then, we washed it thoroughly with water, and it was left to dry in the shade for 15 days. Voucher specimens (NOR-018) were deposited to the Herbarium of the chemistry laboratory, University of El-Oued.

Essential oil extraction
The aerial parts of the plant were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The oil obtained was collected and dried over anhydrous sodium sulfate Na 2 SO 4 to remove moisture. Then, it was stored at 4 °C in sealed brown vials until analysis. The oil analysis was carried out using GC-MS.

Gas chromatography-mass spectrometry (GC-MS) analysis
Mass spectra were recorded using Shimadzu GCMS-QP2010 (Tokyo, Japan) equipped with Rtx-5MS fused bonded column (30 m x 0.25 mm i.d. x 0.25 µm film thickness) (Restek, USA) equipped with a split-splitless injector. The capillary column was directly coupled to a quadrupole mass spectrometer (SSQ 7000; Thermo-Finnigan, Bremen, Germany). The initial column temperature was kept at 45 °C for 2 min (isothermal) and programmed to 300 °C at a rate of 5 °C/min, and kept constant at 300 °C for 5 min (isothermal). The injector temperature was 250 °C. Helium carrier gas flow rate was 1.41 mL/min. All mass spectra were recorded applying the following conditions: (equipment current) filament emission current, 60 mA; ionization voltage, 70 eV; ion source, 200 °C. Diluted samples (1% v/v) were injected with split mode (split ratio 1: 15).

Compounds identification
The determination of oil components was based on comparing their spectra with those of references in the MS Library (NIST and Wiley). To confirm the identification, the corresponding retention indexes (RI) were calculated using the n-alkanes series, C 8 -C 28 , and then compared with those reported in the literature [9]. The relative amounts of individual components were calculated based on GC-FID peak area.

Cytotoxic effect on cancerous cell lines
The cytotoxic activity test (in vitro bioassay on human tumor cell lines) was conducted and determined by the Bioassay-Cell Culture Laboratory, National Research Centre, El-Tahrir St., Dokki, Cairo 12622, Egypt. In this study, we evaluated the cell viability by the mitochondrial dependent reduction of yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan [10]. It is to note that the following procedures were conducted in an aseptic zone using a laminar flow room biosafety class II level (Baker, SG403INT, Sanford, ME, USA). The cells were suspended in RPMI 1640 medium (for HePG2 and HCT116), 1% antibiotic-antimycotic mixture (10,000U/mL potassium penicillin, 10,000 µg/mL streptomycin sulfate and 25 µg/mL Amphotericin B) and 1% L-glutamine at 37 °C under 5% CO 2 . Concentration of 10 x 10 3 cells/well in complete and fresh culture medium in 96-well plastic microtiter plates at 37 °C for 24 hours under 5% CO 2 , using a carbon dioxide incubator (Sheldon, TC2323), Cornelius, OR, USA. The medium was aspirated, the fresh one (no serum) was added and the cells were incubated either alone (negative control) or with different sample concentrations to give a final concentration of (-0.78-1.56-3.125-6.25-12.5-25-50-100) μg/mL. After 48 h of incubation, the medium was aspirated, 40 μL of MTT salt (2.5 μg/mL) was added to each well and incubated for a further four hours at 37 °C under 5% CO 2 . To stop the reaction and dissolve the formed crystals, 200 μL of 10% sodium dodecyl sulfate (SDS) in deionized water were added to each well and incubated overnight at 37 °C. A positive control consisting of 100 μg/mL was used as a known natural cytotoxic agent, giving 100% lethality under the same conditions [11,12]. The absorbance was then measured using a multi-well microplate reader (Bio-Rad Laboratories Inc., Model 3350, and Hercules, California, USA) at 595 nm and a reference wavelength of 620 nm. Statistical significance was tested between the samples and the negative control (cells with vehicle) using an independent t-test by the SPSS 11 program. DMSO is the vehicle used for the dissolution of the plant extracts, and its final concentration in the cells was less than 0.2%. The percentage change in viability was calculated using the following formula (1): Cell viability (%) = (Extract reading/Negative control reading) x 100. (1) A probit analysis was carried out to determine IC 50 and IC 90 using the SPSS 11 program.

Antioxidant capacity Cyclic voltammetry method
The determination of antioxidant capacity was based on cyclic voltammetry (CV) experiments were performed using PGZ402 potentiostat/galvanostat (radiometer analytical SAS) controlled with the VoltaMaster4 software version 7.08 (radiometer analytical SAS). Experiments were realized in a double walled electrochemical cell of 25 mL, and conventional three electrodes system was employed. We used a glassy carbon (GC) working electrode (radiometer analytical SAS), with an area of 0.013 cm 2 , a platinum wire counter electrode, and an Hg/Hg 2 Cl 2 reference electrode (3.0 M KCl). Prior to the voltammetric experiments, the glassy carbon electrode was cleaned with cotton soaked in acetone for 2 min, and then with cotton soaked in water for several seconds. A toluene-ethanolic (50% ethanol, v/v) was used as solvent and 10 µL sulfuric acid as supporting electrolyte. All solutions were prepared using absolute ethanol. Once we cleaned the GC electrode, the required sample volume (of α-tocopherol solution or Ammodaucus leucotrichus oil) was added by micro-pipette into the supporting electrolyte in the electrochemical cell. The cyclic voltammograms were immediately recorded to minimize adsorption (or avoid any other electrochemically active species) onto the GC electrode surface. The cyclic voltammetric conditions used were: scans ranged from 0 to +1 V; scan rate of 100 mV.s -1 . All measurements were done in triplicate.

DPPH radical scavenging activity
The estimation of the remaining DPPH radical concentration in DPPH solution after reaction with essential oils was performed using slightly modified procedure [13,14]. Pure methanol was used to adjust the zero of the spectrophotometer. Methanolic solution of the examined essential oil (1 mL) was mixed with DPPH solution (1 mL) in a 5 mL test tube. The mixture was stirred vigorously for 30 s, poured into the optical cuvettes (1 cm × 1 cm × 3.5 cm) and immediately placed in a spectrophotometer. The changes of absorbance at 515 nm were monitored in a continuous manner during 30 min. The percent of inhibition I (%) was calculated from the formula (2): where A 0 is the absorbance of the control (containing all reagents except the test compound) and A s is the absorbance of tested sample. The oil concentration providing 50% inhibition (IC 50 ) was calculated from the graph by plotting inhibition % against oil concentration. α-Tocopherol was used as reference.

Antimicrobial activity
The  [15] and the volume was adjusted to 10 mL with ethanol (5%, w/v) to obtain bacterial inoculums adjusted to a concentration of 10 6 CFU/mL, which were incubated under shaking conditions (100-120 rpm) for 24 h at 37 °C. Each test was performed in triplicate; on three separate experiments.

Chemical composition
The hydrodistillation of A. leucotrichus areal parts gave blue oil with yield of 1.8%. A total of 24 components were identified and presented about 99.91% of the total detected constituents. The essential oil contained (Table 1) 64.8% of oxygenated monoterpenes and 34.86% of monoterpenes hydrocarbons. It could be noticed that the principal constituent was perillaldehyde (64.66%), followed by D-limonene (26.99%) and the less percentage was attributed to α-pinene (5.8%). At very low concentrations (less than 1%) of other compounds were identified such as; β-pinene 0.66%, β-ocimene 0.65% and camphene 0.4%. Methyl perillate and bornyl acetate were detected in a limited amount of 0.08%. Our results are in agreement with those reported in the literature [14,17], except some differences related to the quantification of the essential oil major components as well as the nature of some identified constituents. For example, according to the studies of [3,17], they both indicated the presence of only a small amount of α-pinene, which disagree with our results [3]. In addition, indicated that Moroccan A. leucotrichus essential oil is constituted mainly of perillaldehyde 88.7% and limonene 8.26%, compared with our results this species contained more perillaldehyde and fewer limonene. In addition further studies reported that A. leucotrichus essential oil extracted from seeds collected in Algeria, contained perillaldehyde 59.12% and limonene 23.89%. Hence the oil originator from Algeria (El-Oued region) is the source of active component (perillaldehyde) used in generally in perfume, cosmetics and for its aroma [18].

Antimicrobial activity
In vitro investigation of A. leucotrichus essential oil was carried out to evaluate its biological efficiency against certain micro-organisms; Bacillus subtilis, Micrococus luteus and Lesteria monocytogene (Gram positive bacteria), Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Agrobacterium tumefaciens and Salmonella enterica (Gram negative bacteria), Mucor ramannianus (fungus) and Candida albicans (yeast). The corresponding minimum inhibitory concentrations (MICs (µL/mL)) of various micro-organisms are shown in (Table 2). The results demonstrated that essential oil under investigation possessed antimicrobial activities against the used micro-organisms.
The different results of the antimicrobial activity of this essential oil by the MIC method are shown in (Table 2). The latter method gives more reliable results compared to the disk method but requires, however, a large oil to test [19,20] . We can globally note the presence of an antimicrobial activity vis-à-vis the essential oil of A. leucotrichus.
With regard to Gram-negative bacteria; Salmonella enterica and E. coli are the most sensitive with a lowest MIC of 5 μL/mL. Of all Gram-negative bacteria, only Pseudomonas aeroginosa gives the highest MIC value while Micrococus luteus is the most resistant gram-positive bacteria with a MIC of 15μL/mL. The same table also shows that the yeast and the fungi manifest in the same manner, giving a MIC of 10 μL/mL. These results are in agreement with those given by [21,8] Finally, the botanical origin of the species seems to play a key role in its antimicrobial efficacy, which confirms the close relationship that exists between the chemical structures of the essential oil constituents.

Cytotoxic activity test
The cytotoxic activity of A. leucotrichus essential oil obtained by hydro-distillation was tested against the two-cell line as it happens HCT116 (colon cell line) and HePG2 (Human hepatocellular carcinoma cell line) [21]. The evaluation of cytotoxicity was performed for essential oil samples concentration that ranged between 0.78 to 100 μg/mL using MTT assays. The results are shown in (table 3). The essential oil possesses cytotoxic activity on HCT116 [21]. Hence the LC 50 (lethal concentration of essential oil which causes the death of 50% of cells in 48 h) and LC 90 (lethal concentration of essential oil which causes the death of 90% of cells in 48 h) was found to be 72.6 μg/mL and 110.6 μg/mL, respectively. The inhibition percentage of this essential oil against HCT116 colon cancer cell line is 78.2% for 100 μg/mL. In fact, the results are very promising compared with positive control Adrinamycin (Doxorubicin) 37.6 μg/mL, while the tested oil is almost ineffective against the HePG2 cell line, for 100 μg/mL the inhibition percentage is 11.5%. Finally, the active compound in oil of A. leucotrichus for its significant cytotoxic activity especially for colon cancer is most likely the perillaldehyde and limonene, acting alone or synergetically with one or more components.

Antioxidant capacity Cyclic voltammetry method
The CV method is actually based on the correlation between the area under the anodic curve of the voltammogram and the electroactivity of the compound under investigation [22]. To calculate the antioxidant capacity, we plotted the calibration curve ( Figure 1) using the voltammogram area versus the concentrations of the α-tocopherol (ranged between 0-450 mg/L) used as standard. From the (Figure 1), we deduced the antioxidant capacity of Ammodaucus leucotrichus oil.
They were next applied to express the total antioxidant capacity of Ammodaucus leucotrichus oil sample as α-tocopherol equivalent. The area under peak current of the background signal (solvent and supporting electrolyte) was subtracted from the area under peak current obtained for sample and α-tocopherol which were recorded within the range of 0 to +1 V. The cyclic voltammogram of Ammodaucus leucotrichus oil sample clearly shows the antioxidant capacity estimated at a value of 47.84 mg α-TE/L. The obtained results are significant, but less important than the antioxidant capacity showed by Origanum majorana and O. vulgare plant oils in previous studies [13]. Table 3. Cytotoxic activity of Ammodaucus leucotrichus essential oil against human cell lines. * Each value represents the mean percentage of inhibition cells of three replicates ±SEM (standard error mean). Ammodaucus leucotrichus oil.

DPPH radical scavenging activity
The essential oil of A. leucotrichus was explored for antioxidant activity using DPPH free radical scavenging power characterized by IC 50 value. Table 4 show the percentage of DPPH inhibition of studied essential oil and α-tocopherol as positive control measured in DPPH assay at different concentrations respectively. The essential oil exhibited a less significant scavenging effect IC 50 = 74.53 mg/mL compared to α-tocopherol (26.426 µg/mL) used as positive control. The tested essential oil (Table 1) is markedly rich in oxygenated monoterpenes (64.8%) which may act as radical scavenging agents [23]. Table 4. DPPH radical scavenging Activity of standard solution (vitamin E).

CONCLUSION
The present work pointed out the investigation of the chemical composition of the essential oil of A. leucotrichus growing in South Algeria on the basis of GC-MS analyzes. The results showed particularly that this oil is rich in perillaldehyde and Dlimonene, added to this; many other compounds were identified and characterized. The in vitro antimicrobial activity of the essential oil was tested on eight strains, one yeast and one fungus. The test showed interesting antimicrobial properties, especially on Salmonella enterica and E. coli. Moreover, A. leucotrichus essential oil exhibited significant antioxidant effects confirmed by two different methods as well as a notable anti-cancer activity with respect to the HCT116 colon cancer cell line.