Phytochemical Characterization, In-Vitro Cytotoxic and Antibacterial Activity of Cotula cinerea (Delile) Vis Essential Oil

Cotula cinerea is a traditional Algerian medicinal plant that has many biological properties, such as analgesic, antiseptic and antibacterial activities. Essential oil of Cotula cinerea was subjected to chromatographical and spectroscopical studies to determine the chemical composition. The analysis highlighted that the oil contains trans thujone (51.86%), santalina triéne (10.6%), αpinéne (2.02%), sabinene (6.17%), cineole <1.8> (5.34%), δ terpinene (1.57%), camphor (2.63%), βterpineol (1.39 %) and terpin-4-ol (1.73%) as the major constituents. The essential oil was tested for antibacterial activity. The oil exhibited substantial antibacterial activity against both the tested Gram negative and Gram positive bacteria. In search of better anticancer agent the essential oil of Cotula cinerea was also subjected for in-vitro cytotoxic activity on two cancerous human cell lines; colon (HCT116) and hepatic (HePG2) cancer cell lines. The results indicates that the essential oil of Cotula cinerea has a significant cytotoxic activity on the tested colon cancer cell lines with a 66.9% inhibition and a minimal inhibition (33.9%) on liver cancer cell line.


Introduction
For better knowing the phytochemical and microbiological properties of the plants of "El-Oued" region, Cotula cinerea [Syn. Brocchia cinerea (Del) Vis], locally known as (Shihia/Shihit El Ebel) 1 , belonging to the family "Asteraceae" was chosen; as it is among the plants mostly used by the local population for TS medicinal properties. Cotula cinerea is used against insolation, colic, cough and bronchopulmonary cooling. This species is widely used in traditional Moroccan medicine for its biological properties such as anti-inflammatory, analgesic, antiseptic, antibacterial, antipyretic activities 2 . It is also used as an infusion to facilitate digestion 3 . The species Cotula cinerea is one of three species belonging to the genus Cotula (Asteraceae) existing in South-Algeria 3 . It is a woolly whitish plant, with thick leaf divided in their upper part to a 3-5 obtuse teeth, stems are 10-40 cm, slept then raised; capitulum from 6 to 10 mm in diameter, woolly involucres with a tubular flower, and brown buds which would become golden yellow 4,5 . Its stems are diffuse or erect. The leaves and whitish-green stems are covered with tiny hairs thick 6 , velvety small leaves, whole are cut into three to seven teeth or "fingers" 7 , and the shaft of high branch yellow inflorescences 8 . The terpenes in the essential oils present are responsible for the characteristic odor 8 . The literature studies highlights that the leaf extracts of Cotula cinerea Del are effective against pathogenic fungi, and also have insecticidal activity on the larvae 9 . It finds importance in the management of stomach pain, fever, headache, migraine, cough and joint inflammation 10 .
In the present investigation, the chemical components of Cotula cinerea Del essential oil was analyzed and screened for antibacterial activity against some pathogenic and food-borne bacteria. The essential oil was also evaluated for anticancer activity against HePG2 (Hepatic) and HCT116 (Colon) human cancerous cell lines.

Plant Material
Aerial parts of Cotula cinerea Del., were collected from the Hassi Khalifa City in the wilaya of Eloued, north east of Algeria during the period of December 2015 to January 2016 for the investigation. Voucher specimens were deposited to the Herbarium of the Chemistry Laboratory, University of El-Oued.

Isolation of the Essential Oils
The aerial parts (100 g) were washed, sorted and dried for a month at room temperature, in the shade and then were finely pulverized by using a mill blade. Clevenger-type apparatus was used for extraction, and hydrodistillation was performed for 4 h 11 . The distilled essential oil was dried over anhydrous sodium sulfate, filtered and stored at 4°C.

Gas Chromatography -Mass Spectrometry (GC/MS) Analysis Conditions
The oil was analyzed by GC on a Hewlett Packard GC-MS system, Model 6890 equipped with Flame Ionization Detector (FID), HP-5, 30m × 0.25mm ID, 0.25 mm film thickness, fused capillary column. The carrier gas was nitrogen (0.8mL/min). Temperature programming was done from 60-250˚C at 4˚C /min with initial and final hold time of 8 minutes. The injection volume was 0.4 μL neat and Split ratio was 1:20. The percentage of the constituents was calculated by electronic integration of FID peak areas without the use of response factor correction and the sample indices were calculated following Van den Dool and Kratz 12 .
The analysis of the volatile constituent was run on a Hewlett-Packard GC-MS system Hewlet Packard 5973 A, equipped with an non polar capillary column HP5MS, 30 m × 0.25 mm, phase thickness 0.25 μm. The detection of elutent was done using an electron ionization system with ionization current: 70eV. Inert helium gas (99.999%) was used as a carrier gas at a constant flow rate of 0.7 mL/min. The injector temperature was 60 and 280°C, respectively. The temperature programmed for oven was from 2°C/min then held at isothermal condition for 8 min and finally raised to 280°C at 2°C/min, split ratio of 1:20, and volume injected was 0.1 to 0.2 μl of the isolated essential oil 13 .

Identification of Compounds
The identification of the essential oil constituents was based on a comparison of their retention times to n-alkenes function (C 9 -C 28 ) 14 . The identification of the oil components were based on MS search Data Library (Wiley & Nist) or with authentic compounds or with the data published in the literature, and Retention Indices (RI) (Adams, 2010) 15 . The chromatographic conditions were identical to those used for GC-MS analysis 16 .

Antibacterial Activity
The  18 and the volume was adjusted to 10 mL with ethanol (5%, w/v) to obtain with 10 μl bacterial inoculums adjusted a concentration of 106 CFU/ mL 17,18 . They were incubated under shaking conditions (100-120 rpm) for 24 h at 37 °C 19,20 .

Antibacterial Activity
The antibacterial activity of the Cotula cinerea essential oil was tested against three Gram positive and five Gram negative pathogenic bacterial strains. The results of the antibacterial activity are shown in Table 2.

In-Vitro Cytotoxic Activity
The cytotoxic activity of the Cotula cinerea essential oil was tested against two cell line HCT116 and HePG2. The results are as shown in Table 3.
The essential oil showed potent cytotoxic activity on HcT116 colon cancer cell line with a 66.9 % inhibition at 100 µg/mL concentration (LC50 86.7 µg/mL and LC90 122.3 ug/mL). The result is very promising compared with positive control doxorubicin 37.6 µg/mL, while the results indicated week activity on HePG2 (33.9 %) inhibition.
Cotula cinerea has also been reported for anticandidal activity 26 . Medicinal plants rich with volatile oil represent an important source of antioxidant and anticancer drugs 27 . The use of essential oil in combination with cancer therapy decreases the side effect of drugs 28 . Essential oil of Ricinus communis containing thujone and 1.8-cinole has been reported for antiproliferative activity 29 . Cotula cinerea showed potent cytotoxic activity especially for colon cancer and this may also be corresponding to the chemical composition of essential oil which is rich in trans thujone 50% and santolina triene, as they are reported for anticancer activity.

Conclusion
The present study reveals antibacterial potency of essential oil of Cotula cinerea of eastern Algeria. C. cinerea can also be an inexpensive source of natural antibacterial substance for use in pathogenic systems to prevent the growth of bacteria and extend the shelf life of the processed foods. C. cinerea showed better cytotoxic activity against colon cancer cell line (HCT116) than liver cancer cell line (HePG2).