Antimicrobial and antioxidant activities of Cymbopogon schoenanthus ( L . ) spreng . essential oil , growing in Illizi-Algeria

Hydrodistilled volatile oil obtained from the aerial parts of Cymbopogon schoenanthus cultivated near Illizi, Algeria, was analyzed by Gas Chromatography Mass Spectrometry (GC-MS) and Gas Chromatography – Flame Ionization Detector (GC-FID). More than twenty compounds were identified, representing 94.636% of the total oil. The major constituents of essential oil were piperitone (63.35%), βeudesmol (9.305%) and elemol (6.915%). Isolated essential oil was tested for radical-scavenging ability using the stable 2,2-diphenylpicrylhydrazyl (DPPH) radical, the 2,2'-azino-bis (ABTS) radical and for reducing power ability with a test based on the reduction of ferric cations (FRAP). In all tests, oil did not show a prominent antioxidant activity. The screening of antimicrobial activity of oil was individually evaluated against representatives of gram-positive, gram-negative bacteria and fungi, using the agar diffusion method. All tested microorganisms were inhibited by the essential oil of C. schoenanthus.


INTRODUCTION
The use of plants for treating diseases is as old as the human species.Popular observations on the use and efficacy of medicinal plants significantly contribute to the disclosure of their therapeutic properties, so that they are frequently prescribed, even if their chemical constituents are not always completely known.Active compounds produced during secondary vegetal metabolism are usually responsible for the biological properties of some plant species used throughout the globe for various purposes, including treatment of infectious diseases.Currently, data on the antimicrobial activity of numerous plants, so far considered empirical, have been scientifically confirmed (Silva et al., 2010).
In Sahara of Algeria, the flora is very rich in medicinal plants which produce valuable natural substances such as essential oil.Actually, essential oil and their components are gaining increasing interest because of their relatively safe status, their wide acceptance by consumers, and their exploitation for potential multipurpose functional use (Boukhris et al., 2012).Many essential oils also have been confirmed to possess the antioxidant activity (Zhang et al., 2006).Natural antioxidants are also in high demand for application as nutriceuticals as well as food additive because of consumer preferences (Neffati et al., 2009).
As part of the study evaluation of the biological effectiveness of the essential oil from the medicinal plants, the study presented a study of the antioxidant and antibacterial activities associated with the chemical composition of essential oil isolated from Cymbopogon schoenanthus (Poaceae).This plant is a subspontaneous grass, tropical-afro-asiatic, which is used as traditional medicines to treat digestive diseases: aerophagia, flatulence and urinary decrease, analeptic drink for new mother after childbirth, bad breath, gumboils and urinary incontinence (Hammiche et al., 2006).

Plant material
The aerial parts of C. schoenanthus were collected from weddi tasset in Illlizi (Algeria), on April, 2014.The vernacular name of this plant is lemmad (the Arabic name) or tiberrimt (the Berber name).Samples of the plants were identified by Dr. Amar Eddoud department of Agricultural Sciences, university of Kasdi Merbah-Ouargla, Algeria

Essential oil extraction
The fresh aerial parts of C. schoenanthus were subjected to hydrodistillation in a modified Clevenger-type apparatus for 3 h.The essential oil was obtained with 2.695 % (w/w) of yield and was dried over anhydrous sodium sulfate and stored in sealed glass vials at 4 to 6°C prior to analyses.

Essential oil chromatographic analysis
0.2 µL of sample was injected on a gas chromatography (Hewlettpackar computerized system, Agilent model 6890 GC coupled to a Agilent 5973 N mass selective detector , and equipped with an Agilent technologies capillary HP-5MS column ( 30 m, 0,25 mm I.d, 0.25µm thickness), a split/splitless injector used in the split mode (200:1), using Helium (20 mL/min).The initial temperature of the column was 40°C which was heated gradually to 325°C with a 2°C /min.High purity helium was used as carrier gas at 36 cm/s.The quadrupole, source and transfer line temperatures were maintained at 150, 230 and 280°C, respectively.A solvent delay of 3 min.Identification of components was assigned by matching their mass spectra of peaks with those obtained from authentic samples and/or the Wiley and NIST library data, and published data (Hellai and Hadj-Mahammed, 2008).The component concentration was obtained by semi-quantification by peak area integration from GC peaks.

DPPH assay
The scavenging activity of DPPH • was determined based on the tests described by Brand-Williams et al. (1995), with some modifications.Various concentrations of essential oil in methanol (1mL) were mixed with methanolic solution containing DPPH radicals (0.1mM).
After vigorous agitation, the mixture was incubated for 1 h in the dark at room temperature, and then the absorbance is measured at 515 nm with a UV spectrophotometer screws (JASCO-V530).A solution containing 1 mL methanol and 2 mL of DPPH radicals was used as blank.The estimation of the antiradical activity is expressed by the value of the percent inhibition (IC (%)) according to the following formula: Where A 0 is the absorbance of analytical blank and A x is absorbance in the presence of the extract solution.
Different sample concentrations were used in order to obtain antiradical curves for calculating the IC 50 (= EC 50 , the effective concentration) values.Antiradical curves were plotted referring to concentration on the x axis and their relative scavenging capacity on the y axis.The IC 50 value, defined as the concentration of antioxidant that causes a 50% decrease in the DPPH • absorbance or the extract concentration providing 50% inhibition.A lower IC 50 value indicates greater antioxidant activity.Evaluation of free radical-scavenging activity was performed with Trolox equivalent antioxidant capacity (TEAC) assay (Neffati et al., 2009;Ćavar et al., 2012;Chen et al., 2013).

ABTS assay
The free-radical scavenging capacity was measured using the ABTS decoloration method (Re et al., 1999) with some modifications.Briefly, ABTS was dissolved in water to get a 7 mM concentration.ABTS radical (ABTS .+ ) was produced by reacting this stock solution with a 2.45 mM K 2 S 2 O 8 solution and allowing the mixture to stand in the dark at room temperature for 12 to 16 h.The ABTS .+solution obtained was blue-green coloration which can be stored at -20°C.Before use, the formed solution was diluted with methanol to an absorbance of 0.70±0.02at 734 nm.Samples were separately dissolved in methanol.In order to measure the antioxidant activity of essential oils, 10 µL of each sample at various concentrations was added to 990 µL of diluted ABTS .+ .The absorbance was measured spectrophotometrically at 734 nm using a UV spectrophotometer screws (JASCO-V530).Methanol was used to zero the spectrophotometer; ABTS .+solution was used as blank sample.The radical-scavenging activity of the tested samples, expressed as percentage inhibition of ABTS .+(IC (%)), were calculated according to the formula IC (%) = [(A 0 −A x )/A 0 ]×100, A x and A o were the absorbance at 734 nm of samples with and without essential oils, respectively.
IC 50 values, defined as the inhibiting concentrations of substrate that causes 50% loss of ABTS activity (color), were calculated by regression analysis.A lower IC 50 value indicates greater antioxidant activity.Evaluation of free radical-scavenging activity was performed with Trolox equivalent antioxidant capacity (TEAC) assay (Neffati et al., 2009;Ćavar et al., 2012;Chen et al., 2013).

FRAP assay
This method measures the ability of antioxidants to reduce the ferric iron.It is based on the reduction of the complex of ferric iron and 2,3,5-triphenyl-1,3,4-triaza-2 azoniacyclopenta-1,4-diene (TPTZ) in the ferrous form under acidic conditions.The reducing power is determined by the method described by Binsan et al. (2008).The FRAP reagent was freshly prepared by mixing acetate buffer (300 mM, pH 3.6), TPTZ solution (10 mM TPTZ in 40 mM HCl) and FeCl 3 •6H2O (20 mM) in a ratio of 10:1:1 (Benzie and Strain, 1996).The FRAP solution was incubated at 37°C for 30 min.In a volume of 150 µL were prepared different concentrations of the sample to which was added 2850 μL of FRAP solution, incubated 30 min in the dark.Complex formation ferrous tripyridyltriazine (colored product) is measured by reading absorbance at 593 nm.The activity is expressed as Trolox equivalent (micromoles TE/g of extract).

Calculation of Trolox equivalent antioxidant capacity (TEAC)
The free radical-scavenging activity of each sample was expressed as Trolox equivalent antioxidant capacity (TEAC), which was obtained by comparing the absorbance change at 515 nm for DPPH essay, at 734 nm for ABTS essay and at 593 nm for FRAP essay, in a reaction mixture containing a sample of plant extract or test material with that containing Trolox.This index is defined as the millimolar concentration of a Trolox solution whose antioxidant capacity is equivalent to 1.0 mg of the extract (Re et al., 1999).

Antimicrobial screening
The antimicrobial activities were determined by using the drop agar diffusion method (Lopes-Lutz et al., 2008).The microorganisms tested were the fungi Candida albicans ATCC 10231, and the bacteria Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 14028, Staphylococus aureus ATCC 6538, Enterococcus feacium ATCC 19434, and Streptocoque B (Streptococcus agalactiae).The oils were diluted in 10% DMSO/sterile H 2 O solution.A suspension of the tested microorganisms was spread on the appropriate solid media plates and incubated overnight at 37°C (for the bacteria) or 25°C (for conidia of filamentous fungi).After 1 day, 4-5 loops of pure colonies were transferred to saline solution in a test tube for each bacterial strain and adjusted to the 0.5 McFarland turbidity standard (~10 8 cells/mL) (National Committee for Clinical Laboratory Standards (NCCLS), 1999).Sterile cotton dipped into the bacterial suspension and the agar plates were streaked three times, each time turning the plate at a 60° angle and finally rubbing the swab through the edge of the plate.Sterile paper discs (Glass Microfibre filters, Whatman; 6 mm in diameter) were placed onto inoculated plates and impregnated with the diluted solutions (15μL/ disc).Ampicillin (10μg/disc) was used as positive control for all strains except C. albicans for which Nystatin (100μg/disc) was used.Inoculated plates with discs were placed in a 37°C (or 25°C for conidia) incubator.After 24 h of incubation, the results were recorded by measuring the zones of growth inhibition surrounding the disc.Clear inhibition zones around the discs indicated the presence of antimicrobial activity.The test was run in duplicate.

Chemical composition of essential oil
A total of 35 compounds were detected in the study essential oil extracted from C. schoenantus but just 24 compounds were identified which accounted for 94.636 % of the essential oil (Table 1).The major component was piperitone (63.35%), the dominant compounds in lemmed oil were oxygenated monoterpenes (65.361 %) followed by oxygenated sesquiterpenes, monoterpens hydrocarbons and sesquiterpenes hydrocarbons.The same compounds have been obtained in a work done previously, when the study used the microwave-assisted hydrodistillation method to extract the essential oil of C. schoenantus in order to determine the impact of the extraction method on volatile constituents was used, 33 compounds were identified in this sample, the most abundant components were oxygenated monoterpenes (74.6 %) with high content of piperitone (72.6 %), monoterpens hydrocarbons (11.30%) followed by oxygenated sesquiterpenes (9.79 %) and sesquiterpenes hydrocarbons (1.48 %) (Hellalli et al., unpublished data).
The literature review showed variation between chemical compositions of essential oil of C. schoenantus, depending on geographical origin.Due to the numerous published articles about chemical composition of C. schoenantus essential oil, in this section the recent findings will be discussed.The essential oil of the Togolese C. schoenantus (Ketoh et al., 2005(Ketoh et al., , 2006;;Koba et al., 2004) is characterized with high content of piperitone (61.01 to 69.01%) and the 2-carene (16.48 to 23.4%).While same species cultivated in Burkina Faso was not very rich in piperitone (42%) and the 2-carene (8.2%), but there is a significant presence of elemol (6.2%) (Onadja et al., 2007).
As a result the study can say that the essential oil obtained from C. schoenantus showed significant variation in their chemical composition depending on geographical origin.Generally, C. schoenantus essential oils were characterized by high percentages of the monoterpenenoid compounds, such as piperitone, 2carene and limonene.

Antioxidant activities
The antioxidant activity of C. schoenantus essential oil has been evaluated by three testing methods ABTS, DPPH and reducing power, the results is summarized in Table 2.It was found that the essential oil showed very different antioxidant capacities.The DPPH assay is most extensively used to evaluate antioxidant activity of plant extracts, foods and single compounds, thanks to its stability and its easiness.This assay is based on the measurement of the reducing ability of antioxidants toward DPPH radical, through electron spin resonance (EPR) detection or by measuring the decrease of its absorbance.DPPH is a stable and commercially available organic nitrogen radical, which reacts with hydrogen/electron donor compounds and has a maximum UV-V is absorption within the range of 515 to 520 nm.Upon reduction, the radical solution becomes discoloured according to the number of electrons paired (Chen et al., 2013).
The estimate of the anti-radical activity is based on a colorimetric test based on the measurement of the relative ability of an extract to trap preformed radical  ABTS .+ .The latter is generated by the oxidation of ABTS (2,2'-azinobis-acid (3-ethylbenzthiazoline-6-sulfonic acid) with potassium persulphate giving a blue-green solution.
The radical ABTS .+ is reduced in the presence of an antioxidant electron donor compound (Re et al., 1999).This reduction results in a proportional bleach percent inhibition of the chromophore ABTS .+and depending on the concentration of the antioxidant.These measurements are compared to the reactivity of a reference compound, generally Trolox.This defines the Trolox equivalent antioxidant capacity (TEAC).The ABTS method is used to study the activity of the compounds hydrophilic and lipophilic antioxidants, pure compounds and extracts for food (Re et al., 1999).The total antioxidant potential of a sample was determined using the ferric reducing ability of plasma (FRAP, also Ferric ion reducing antioxidant power), which was first performed by Benzie and Strain (1996), as a measure of antioxidant power.The FRAP assay measures the change in absorbance at 593 nm owing to the formation of a blue colored ferrous tripyridyltriazine (Fe II -tripyridyltriazine) compound from the colorless oxidized Fe III form by the action of electron donating antioxidants (Politeo et al., 2006).
Comparing IC 50 and TEAC values obtained (Table 2) the C. schoenantus essential oil showed an antioxidant activity in the ABTS essay most important than in the DPPH essay, although DPPH and ABTS methods were based on the same principle, data obtained from ABTS assay are lower than those obtained from DPPH assay (Ćavar et al., 2012).In order to compare results given earlier, the study tested the ability of C. schoenantus essential oil of the reducing power of the ferric ion (F +3 ) to corresponding ferrous ions (F +2 ).
In general, the antioxidative effectiveness of essential oil depends on the content of phenolic compounds and the reaction activity of the phenol towards the chaincarrying peroxyl radicals and on the stability of the phenoxyl radical formed in the reaction (Ćavar et al., 2012).The essential oil obtained from C. schoenantus growing in Illizi is markedly rich in non-phenolic constituents, due to this fact the essential oil is known to be relatively weak antioxidant.

Antimicrobial activity
The antimicrobial activity of C. schoenantus essential oil was evaluated by a paper disc diffusion method.The data show that the essential oil of C. schoenantus exhibited strongly all the tested strains, but in variable degree.The results presented in Table 3, reveal that C. schoenantus essential oil inhibited strongly the growth of E. faecium and S. aureus.The study used antibiotic ampicillin as a positive probe.
Generally, the higher resistance among Gram-negative bacteria could be ascribed to the presence of their outer phospholipidic membrane, almost impermeable to lipophilic compounds (Rattanapitigorn et al., 2006;Boukhris et al., 2012).The absence of this barrier in Gram-positive bacteria allows the direct contact of the essential oil hydrophobic constituents with the phospholipids bilayer of the cell membrane, where they bring about their effect, causing either an increase of ion permeability and leakage of vital intracellular constituents, or impairment of the bacteria enzyme (Dorman and Deans, 2000;Burt, 2004;Boukhris et al., 2012).That is way the results indicated that all the Grampositive bacteria were the most sensitive strains tested to the oil of C. schoenantus, especially E. faecium (with dilution ration (1/32).But, it's known that Enterococci are normal commensals of the human intestinal tract and may cause various kinds of infections, including primary bacteremia, endocarditis, meningitis, and urinary tract infection.
E. faecalis and E. faecium account for the vast majority of clinical isolates in human enterococcal infections.These organisms have intrinsic and, often, acquired resistance to a number and high frequency of antibiotics, causing treatment of serious infection to be challenging (Murray, 1990;Arias and Murray, 2012).Tested oil showed a strong antimicrobial activity against all microorganism species, this can explained the effectiveness in the traditional treatment to heal the urinary tract diseases, digestives diseases and to post partum care for the new mother.This allows the study to confirmed the antimicrobial properties in vitro of Cymbopogon schoenantus, from Illizi (Extreme southeast of Algeria), through the effectiveness of its essential oil against the microorganisms tested.The high antimicrobial activity registered of C. schoenantus essential oil, can be explained by the high proportion of piperitone.This component was found in many plants like Mentha, Cymbopogon and Eucalyptus spp.Piperitone isolated from C. schoenantus in Togo was essentially a (+) enantiomer as generally observed in Cymbopogon spp.The good insecticidal properties of piperitone have been already reported.Indeed, piperitone, isolated from the essential oil of Artemisia judaica, has demonstrated a complete antifeedant activity at a concentration of 1000 µg/mL against the third instar larvae of Spodoptera littoralis using non-choice leaf discassay (Abdelgaleil et al., 2008;Bossou et al., 2015).
Furthermore when tested against Callosobruchus maculatus, piperitone, isolated from C. schoenanthus, was more toxic by fumigation to adults with a IC 50 value of1.6 µL/L vs. 2.7 µL/L obtained with the crude extract (Ketoh et al., 2006;Bossou et al., 2015).Piperitone was also reported as a powerful repellent and antiappetent agent against ant of Crematogaster spp.Against C. maculatus, piperitone was very toxic to adults, newly laid eggs and to neonate larvae.But it was less efficient against individuals developing inside the seeds than the crude oil.The high larvicidal activity observed on 5-day old larvae with the crude oil was probably due to other compounds, which can act on various target sites at insect level like monooxygenases or acetylcholinesterases (Ketoh et al., 2006).The antimicrobial activities have been mainly explained through the presence of oxygenated sesquiterpenes and monoterpenes.The synergistic effect of essential oil components is a promising field that could lead to the optimization of a given bioactivity (Alitonou et al., 2012).

Conclusion
To the best of the study knowledge, this is the first study providing data on antibacterial, antifungal and antioxidant activities of the essential oil of C. schoenanthus from Algeria especially from the region of Illizi.Their antibacterial and antioxidant activities was studied in vitro on five bacterial strains and one fungal strain, all microbial strains were inhibited by the oil obtained from C. schoenanthus, but it strongly inhibits the growth of Enterococcus faecium.C. schoenanthus essential oil showed that it can be considered as a relatively weak antioxidant, this may be due to its lack of phenolic components.

Table 1 .
Chemical composition of essential oil of C. schoenantus.

Table 2 .
Antioxidant activity of C. schoenanthus expressed in TEAC and IC 50.

Table 3 .
Antimicrobial activity of C. schoenantus essential oil.
*Including disc diameter of 6 mm, Averages ± Standard Deviation were obtained from two different experiments; ** The essential oil was diluted in 10% DMSO/sterile H 2 O solution