Chemical composition, antioxidative, antimicrobial and anti-cancer activities of Asteriscus graveolens (Forssk) essential oil

This work is devoted to the study of the chemical composition and the evaluation of the biological activities of essential oils (EOs) extracted from the flowers of Asteriscus graveolens Forssk. plant. The EO sample was obtained by hydrodistillation and the chemical composition analysis was performed using GC and GC/MS. The major chemical components characterizing the EO were cis-chrysanthenyl acetate (44.30%) and cis-8-acetoxychrysanthenyl acetate (33.70%). Antioxidant activity was determined using DPPH and Phosphomolybdenum tests. Although the EO presented a weak scavenging activity (420.16 mg/mL), it exhibited good reducing power using the Phosphomolybdenum assay (0.28 AAEC/mg). The most important antibacterial activity was noted for Bacillus cereus. The oil revealed a remarkable activity against the nine fungi species tested with percentage inhibition up to 94.12% for Fusarium culmorum (BTCR). More important, this work investigated for the first time the anticancer effect of this EO on two types of cancer cell lines (human liver carcinoma and Rat pheochromocytoma cell lines). The EO showed a high anticancer activity against both tumor cell lines comparing to the positive control.


Introduction
The biological properties of natural products as essential oils (EOs) are studied to search a new drugs, antibiotics and pesticides (Buchanan et al. 2000). They have been screened for their potential uses as alternative remedies for the treatment of many infectious diseases and the preservation of food from the toxic effects of oxidants.
With the emergence of antibiotic-resistance, a reliable alternative to antibiotics is the use of EOs. These compounds are known for their antibacterial properties which have been demonstrated in vitro and in vivo (Zhiri 2006). Many works were devoted for the study of biological properties of EOs, thus, the antimicrobial activity of oils extracted from thyme; rosemary and chamomile were demonstrated (Bakkali et al. 2008;Dorman and Deans 2000;Floris et al. 1996;Marino et al. 2001). Because of the possible toxicities of the synthetic antioxidants, increasing attention has been directed toward natural antioxidants (Avlessi et al. 2004). The antioxidant activity of essential oils has been the subject of much intensive research because of their uses as preservatives in food industry even at low concentrations (Cabrera and Prieto 2010).
Fungal plant pathogens cause many damages to food production and storage. They may be at the origin of catastrophes in which large areas planted to food crops are destroyed (Strange and Scott 2005). In order to combat the losses they cause, it is necessary search remedies. Many studies have been devoted to the antifungal activity of EOs and revealed an important effect especially against phytopathogen fungal (Marinelli et al. 2012;Matusinsky et al. 2015;Znini et al. 2011). Cancer belongs to a huge class of diseases, which cause more than 10% of all human deaths. Many studies showed the potential of the constituents of essential oil on several cancer cell lines (Adorjan and Buchbauer 2010).
Asteriscus graveolens Forssk (syn. Nauplius graveolens Forsk, Bubonium graveolens Forsk Maire) belongs to the Asteraceae family of herbs. It's very common in the North Africa. Their leaves are collected in the spring, they are prepared as infusion or decoction; the sap of the fresh leaves is used as drops for the nose and poultice for headaches. Traditionally, it is used for blennorrhagia, diabetes, diarrhea, facial neuralgia, head cold, gastralgia, pulmonary problems and sinusitis (International Union of Conservation of Nature IUCN 2005).
In the present work, we report the results of a study aimed to define chemical composition and to evaluate antioxidant, antibacterial, antifungal and for the first time the anticancer activity of A. graveolens EO.

Plant material and essential oil extraction
The aerial part of A. graveolens was collected from south of Algeria, during the month of June 2013 at the flowering stage. It was identified at the National Institute of Agronomy "INA Institut National d'Agronomie". The separation of the flowers from the aerial parts was executed manually and with care. Then the air drying of the plant material was performed in the shade at room temperature for 10 days. The dried plant sample (flowers) was subjected to hydrodistillation using a Clenvenger type apparatus for 3 h. The distilled EO was dried over anhydrous sodium sulfate and stored at + 4 °C until use.

Gas chromatography and GC/MS analysis
An Agilent technologies 7890A gas chromatograph (GC) equipped with a flame ionization detector (FID) was used for compound separations with a HP5 capillary column (30 m × 0.32 mm, film thickness 0.40 μm). The oven temperature program was as follows: 2 min at 80 °C, from 80 to 200 °C at 5 °C/min, then 5 min at 200 °C, then from 200 to 260 °C at 20 °C/min, then 5 min at 260 °C. Detector and inlet temperatures were 280 °C. Hydrogen was used as carrier gas at a constant flow of 1 mL/min with a split ratio 1:70. A volume of 1 μL of EO diluted in dichloromethane was injected manually. The retention indices of constituents were calculated relative to a series of n-alkane standards n-C 8 -C 26 .
The GC/MS analysis of the EO was carried out on an Agilent HP-6890 coupled to 5973 mass spectrometer and equipped with UB-WAX capillary column (30 m × 0.25 mm, film thickness 0.25 μm) and quadrupole detector (70 eV). The carrier gas flow (Helium) was fixed at 1 mL/min. The transfer line and injector temperatures were fixed at 220 and 250 °C, respectively. 1 µL of diluted EO in ethanol solution (1:100, v/v) was injected manually in a splitless mode.
Identification of components was based on comparison of their mass spectra with those of WILEY and NIST Libraries as well as on comparison of their retention indices with literature.

DPPH assay
In the presence of a proton donor substance, the free radical DPPH takes the non-radical form and loses its purple color (Molyneux 2004). One milliliter of EO dilution in ethanol solvent was added to 1 mL of 200 µM DPPH solution prepared in ethanol solvent. After incubation for 30 min in a dark, the absorbance was measured at 517 nm against a blank (ethanol). Decreasing the absorbance of DPPH indicates an increase in DPPH radical scavenging activity, which is calculated according to the equation: where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.
Control was prepared by a mixture of 1 mL of DPPH solution and 1 mL of ethanol. The sample concentration providing 50% of radical scavenging activity (IC 50 ) was determined through the curves obtained. The lower IC 50 indicates higher radical scavenging activity and vice versa. Ascorbic acid and α-tocopherol were used as standards.

Phosphomolybdenum assay
The reducing power of EO was measured according to the method of Prieto et al. (1999). The principle is based on the reduction of molybdate VI to molybdate V in presence of reducing substance, then the molybdate V form a greencolored complex and the absorbance is measured at 695 nm. An aliquot of 0.2 mL of EO solution in ethanol was added to 2 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The tubes were sealed and incubated in water bath at 70 °C for 90 min. After the incubation period, and when the samples had cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against a blank containing 2 mL of reagent solution and 0.2 mL of ethanol. The antioxidant capacity was expressed as equivalents of ascorbic acid AAEC.

Bacterial strains
For the determination of the antibacterial activity of the EO, we used standard and isolated strains of the following Gram-positive bacteria: Bacillus cereus ATCC 12778, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, MRSA ATCC 43300. The EO was also tested on Gram-negative bacteria: Escherichia coli ATCC 25922, Klebsiella pneumonia ATCC 700603, Pseudomonas aeruginosa ATCC 27853, Salmonella typhi (isolate). The microorganisms were obtained from the culture collection of the "Regional Veterinary Laboratory" and the "Medical Analysis Laboratory of the hospital of Laghouat".

Disc diffusion method
This method was employed to evaluate the antibacterial activity (Bekhechi et al. 2008). All bacterial strains were first grown on nutrient agar plates at 37 °C for 18-24 h, prior to inoculation into the nutrient broth. One hundred micro-liter of bacterial suspension containing 10 8 cell/mL was spread onto Mueller-Hinton culture medium. A sterile filter disc (diameter 6 mm) impregnated with 10 µL of the diluted EO in 10% DMSO (1/4), was placed onto the plate culture. The Petri dishes were stored at 4 °C for 2 h and then incubated for 24 h. The diameters (mm) of the inhibition zones were measured. Each experiment was done in triplicate. Albedinis and Fusarium oxysporum f. sp lycopersici was provided by the "Regional Station of Plant Protection of Ghardaïa" in Algeria. Antifungal activity was studied by using contact assay. The EO dilutions in 2% agar solution was added to the molten PDA medium and poured into Petri dishes (El Ajjouri et al. 2008). The control plate contains PDA added 2% w agar solution. A 6 mm diameter disk of the fungal species was cut from a 1-week-old culture on PDA plates, and then the mycelia surface of the disk was placed upside down on the center of dish.

Antifungal activity
The incubation was performed at 25 °C for 7 days. The Extension Diameter (millimeters) of hyphae from the center to the side of the dish was measured and the inhibition percentage was calculated as follows (Aoudou et al. 2010): where D 0 is an average of 3 replicates of hyphal extension (mm) of controls and D I is an average of 3 replicates of hyphal extension (mm) of plates treated with EO.

Anticancer activity
Cell lines HePG2 (human liver carcinoma) and PC12 (Rat pheochromocytoma cells) were maintained at 37 °C in a 5% CO 2 atmosphere. Both cell lines were grown in a medium containing 5 mL of RPMI-1640 supplemented with 10% fetal bovine serum (FBS).The effect of the EO on cell viability of the two cancer cell lines was determined by 3 [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay (Mosmann 1983). The growing cells were plated in 96-well microplates at a density of 5 × 10 3 cells per well in 100 µL of culture medium and allowed to adhere for 24 h before treatment. Increasing concentrations of EO were then added (100 µL/well). The cells were incubated 48 h in the presence and absence of essential oil. 5-fluorouracil (6.25-100 µg/mL) was used as positive control. 20 µL of MTT (5 mg/mL of PBS) were added to each well, and the cells were incubated for a further 4 h. After removal of the medium, 200 µL DMSO was added to each well. The absorbance was recorded on a micro plate reader at the wavelength of 490 nm. The effect of the essential oil on cell growth inhibition was assessed as percentage cell growth inhibition. This test was replicated five times.

Essential oil composition
Hydrodistillation of A. graveolens flowers yielded 1.16 ± 0.2 mL of yellow essential oil upon 100 g of dry matter.

DPPH assay
The antioxidant capacity of the EO was determined by comparison to the activity of known antioxidants: ascorbic acid and α-tocopherol. The assessed EO was able to reduce the stable, purple colored radical DPPH to the yellowcolored DPPH-H reaching 50% of reduction with an IC 50 of 420.16 mg/mL. Comparison of the IC 50 of investigated EO with those obtained by ascorbic acid and α-tocopherol showed that this EO expressed very weak activity compared to the standards ( Table 2). The weak DPPH radical scavenging activity of this oil could be attributed to the absence of active components. When Alilou et al. (2014) studied the antioxidant activity of A. graveolens ssp. odorus EO using DPPH test, they found an important activity expressed as IC 50 value of 0.2498 mg/mL, except they used the whole aerial part of this plant which certainly should make the difference in this comparison.  1 3

Phosphomolybdenum assay
The results for Phosphomolybdenum assay were presented as equivalents of ascorbic acid per milligram. Higher values indicate better antioxidant activity. The EO showed important value relative to ascorbic acid ( Table 2). The activity of the EO of A. graveolens represented about half (roughly) of α-tocopherol activity. Vidic et al. (2016) used this method to evaluate the antioxidant activity of some Asteraceae species (Achillea millefolium L., Arnica Montana L., Artemisia absinthium L., and Artemisia annua L.) and the highest antioxidant activity using the Phosphomolybdenum assay was obtained for Arnica Montana L. (55.69 mg AAEC/g).

Antibacterial activity
The inhibitory effect of A. graveolens essential oil on the growth of eight bacterial strains was tested. The EO showed various degrees of antibacterial activity depending on the bacterial strain. As showed in Table 3 the most important activity was observed on B. cereus with inhibition diameter of 12.5 ± 0.50 mm. For the other bacterial strains, the inhibition zone was ranging from 9 to 10.33 mm (  (Melekmi et al. 2006). Table 4 shows the mean radial growth of the fungus species on solid medium containing A. graveolens EO. The mycelial growth diameter of each species was measured during 6 days of incubation. The A. graveolens EO exhibited antifungal activity against the phytopathogenic fungi (Table 4). The percentage inhibition of mycelial growth increased with increasing concentration of EO for all strains tested. The most important activity was noted against F. culmorum BTCR with an inhibition growth of 94.12% corresponding to 20 µL/mL of EO. The inhibitory concentration of 50% of growth (IC 50 ) for the species varied between 0.5 and 1.0 µL/mL. This activity could be due to the major component of this EO (cis-chrysanthenyl acetate and   (Terzi et al. 2007).

Antifungal activity
Comparing to the study of Cakir et al. (2005), the EO of Hypericum linarioides had a weak activity on F. culmorum with inhibition percentage of 13.6% at 5 mg/mL. In another study the Elettaria cardamomum EO had an important activity against F. graminearum (90 ± 1.3% at 3000 ppm) using the inverted Petri dish method (Singh et al. 2008). The EO of A. graveolens aerial parts have a strong activity against Alternaria spp. and moderate activity against penicillium expansum with inhibition rate of 100% and 74.63%, respectively at 0.2% (v/v) of the oil (Znini et al. 2011). The EO of Schinus spp. and Thyme have also a strong activity against F. graminearum (Sampietro et al. 2014).

Anticancer activity
The results for cell growth inhibition by A. graveolens EO against Rat pheochromocytoma (PC12) and human liver carcinoma (HepG2) cell lines for various concentrations are shown in Figs. 1 and 2, respectively. This experiment shows that the EO of A. graveolens has an important activity against PC12 cell line comparing to the positive control. The activity against HepG2 cell line was less important at low concentrations but at 100 µg/mL, 82.98% of cells growth was inhibited. This activity could be due to their major components (cis-chrysanthenyl acetate and cis-8-acetoxychrysanthenyl acetate) or to a synergic with other components. Many species of Asteraceae (Matricaria chamomilla L., Artemisia desertorum L., Artemisia chamaemelifolia Vill.) are known for their anticancer activity (regarding their EOs) (Lesgards et al. 2014).
The concentrations providing 50% of cell growth inhibition were less than 6.26 µg/mL for the PC12 cell line and more than 50 µg/mL for the HepG2. The results obtained by Wang et al. (2012) showed that the human hepatocellular liver carcinoma cell line (Bel-7402) were the most resistant to Rosmarinus officinalis L. EO and its major components among the tested cancer cell lines; however EO from Artemisia indica (Asteraceae) have a strong toxic effect on liver cancer cell lines HepG2 (Rashid et al. 2013).

Conclusion
The chemical composition of A. graveolens (Forssk) flowers essential oil was determined. The results showed two major components: cis-chrysanthenyl acetate and the cis-8-acetoxychrysanthenyl acetate. Moreover, we found for the first time some interesting antifungal and anticancer activities suggesting the presence of very active components in this EO. Further studies to elucidate the mechanisms of action, and the possible compounds involved in these activities will be undertaken.

Compliance with ethical standards
Ethical statement The study was conducted following the approval by the Institutional Animal Ethical Committee of Amar Télidji University, Laghouat, Algeria. Fig. 1 Anticancer activity of A. graveolens flowers essential oil against PC12 cell lines