Chemical composition of Citrus limon ( Eureka variety ) essential oil and evaluation of its antioxidant and antibacterial activities

The study evaluates the chemical composition, antioxidant and antimicrobial effects properties of essential oil of Citrus limon (Eureka) extracted by hydrodistillation. The composition of this oil was analysed by GC/MS for 30 constituents, which accounted for 97.8% of total oil. The main components were limonene (61.3%) followed by β-pinene (9.7%), α-citral (4.2%) and α-terpinene (3.8%). Antioxidant activity of the C. limon essential oil was evaluated by using DPPH radical scavenging and β-carotenelinoleic acid bleaching. In both tests, the oil showed antioxidant property close to that of positive control (α-tocopherol). The essential oil was tested against nine bacteria (two Gram+: Bacillus cereus, Staphylococcus aureus ATCC 29213 and seven Gram-: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica, Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marescens, Proteus mirabilis) by using disc diffusion and microdilution methods. C. limon essential oil showed antimicrobial effect against all microorganisms tested. Maximum activity (MIC = 180 μg.ml -1 ) was observed against Staphylococcus aureus.


INTRODUCTION
Citrus is produced in abundance in several areas worldwide (Thomas and Spreen, 2010).The amount of residue obtained from Citrus fruits account for 50% of the original amount of the whole fruits (Chon and Chon, 1997).Essential oils have diverse and high biological applications.For instance, they are used in the medical field like biocidal activities (bactericidal, virucidal and fungicidal) and medicinal properties (Mayaud et al., 2008).The use of essential oils as food preservatives has been described (Burt, 2004;Tiwari et al., 2009).Because of their complex chemical composition which composed of more than 100 different terpinic compounds, essential oils have a broad biological and antimicrobial activity spectrum (antibacterial, antifungal, antimoulds, antiviral, pest control, insect repellents).
Recently, the essential oils and various extracts of plants have been of great interest as they have been important sources of natural products.In order to prolong the storage stability of foods in industrial processing, synthetic antioxidants and antibacterials are used.Otherwise, side effects of some synthetic conservatives used in food processing have been documented (Ames, 1983;Baardseth, 1989).For this reason, governmental authorities and consumers are concerned about the safety of the food and potential effects of synthetic additives on human health (Reische et al., 1998).
Despite their wide uses and fragrances, essential oil constitutes an effective alternate to synthetic compounds produced by chemical industry without having any side effects (Faixova and Faix, 2008).The objective in this present study is to evaluate the antioxidant and antibacterial properties of essential oil of Citrus limon (Eureka variety) extracted by hydrodistillation.

Process of extraction
The essential oil of C. limon (Eureka) peel is extracted by steam distillation for 2 h and 30 min using a Clevenger-type apparatus.The supernatant was separated by decantation after addition of 50 % NaCl.The essential oil was collected, dried over anhydrous sodium sulfate, and stored in glass vials covered with aluminum foil at 4°C until used.

Gas chromatography-mass spectrometry
The essential oils were analyzed by gas chromatography coupled to mass spectrometry (GC/MS) (using a DB-5 fused-silica-capillary column polar (30 m × 0.25 mm, 0.25 µm film thickness).The oven temperature program was 60°C and held for 8 min, then increased from 45 to 250°C at a rate of 2°C/min which was held at 250°C for 20 min.Helium gas was used as the carrier gas at a constant flow rate of 3 ml/min.Injector and MS transfer line temperatures were set at 250°C and 280°C, respectively.The temperature of electronic impact at 70 eV source was 230°C.Samples (0.2 µl) were injected at 250°C at a split ratio 50:1.Identification of the components was made by determination of their retention indices (KI) relative to homologous series of n-alkanes (C8-C28) (Fluka, Buchs SG, Switzerland) by matching recorded mass spectra with those stored in spectrometer database (NIST MS Library v. 2.0) and bibliography (Adams, 2001).Component relative percentages were calculated based on GC peak areas.

Scavenger effect on DPPH
The ability of the essential oil to donate hydrogen atom or electron Himed et al. 357 and scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was determined by slightly modified method of Brand (Brand-Williams et al., 1995).The concentrations of the tested essential oil ranged from 2 to 0.05 mg/ml.A portion of sample solution (200 µl) was mixed with 3 ml of 5.25 × 10 -5 mol/l DPPH in absolute ethanol.The decreasing absorbance of the tested mixtures was monitored every 1min for 30 min at 515 nm using ultraviolet and visible (UV-Vis) spectrometer.
Absolute ethanol was used to zero the spectrophotometer.The DPPH solution was used as a blank sample and αtocopherol was used as a positive probe.The radical scavenging activity of the tested essential oil, expressed as percentage inhibition of DPPH, was calculated according to the following formula: Where, At is the absorbance value of the tested sample and A0 is the absorbance value of the blank sample, in a particular time (t).The percentage of inhibition was plotted after 30 min against concentration, and the equation for the line was used to obtain the IC50 value.

Β-carotene bleaching assay
Antioxidant capacity is determined by measuring the inhibition of the volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic acid oxidation (Dapkevicius et al., 1998).A mixture of 2 mg β-carotene and 25 µl linoleic acid was prepared in 10 ml of chloroform and 200 mg Tween 40.The chloroform evaporated completely at 40°C under vacuum.50 ml of oxygenated distilled water was subsequently added to the residue and mixed gently to form a yellowish emulsion.The essential oil and α-tocopherol (positive control) were individually dissolved in methanol (2 mg/ml), 350 µl volumes of each were added to 5 ml of the above emulsion in test tubes and mixed thoroughly.The test tubes were incubated in a water bath at 50°C for 2 h together with a negative control (blank) which contained the same volume of methanol instead of the essential oil.The absorbance values were measured at 470 nm on an ultraviolet and visible (UV-Vis) spectrometer.The antioxidant activities (inhibitions percentage, I%) of the samples were calculated using the following equation : Where, Aβ-carotene after 2 h assay is the absorbance value of βcarotene after 2 h assay remaining in the sample and Ainitial β-carotene is the absorbance value of β-carotene at the beginning of the experiments.

Bacterial strains
The essential oil was tested against nine strains of food borne pathogenic bacteria: two Gram+: Bacillus cereus, Staphylococcus aureus ATCC 29213 and seven Gram-: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica, Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marcescens and Proteus mirabilis.Bacterial strains were cultured overnight at 37°C on Mueller Hinton broth and adjusted to a final density of 10 6 CFU/ml, used as an inoculum.

Diffusion assay
In vitro, antibacterial activity of the essential oil was evaluated against the nine bacterial strains by the disk diffusion method (Rota IC (%) = [(A 0 -A t )/A 0 ] × 100 = ( A β-carotene after 2h assay / A initial β-carotene ) x100 et al., 2004).The test was performed in sterile Petri dishes containing solid and sterile Mueller-Hinton agar medium.The essential oil (5 µl) absorbed on sterile paper discs (Whatman disc of 6 mm diameter), were placed on the surface of the media previously inoculated with 100 µl of microbial suspension (10 6 CFU/ml), then the Petri dishes were incubated at 37°C for 24 h after staying at 4°C for 2 h.The inhibition zone diameters around each of the disks (diameter of inhibition zone including the disc diameter) were measured in millimeters.

Determination of minimal inhibitory concentration (MIC)
The minimal inhibition concentration (MIC) values were studied for the bacterial strains which were sensitive to essential oil in disc diffusion assay.Minimal inhibition concentration (MIC) values were determined by broth micro dilution method (Carson and Riley, 1995).The test was performed in Mueller Hinton broth (MHB) supplemented with Tween 80 (concentration of 0.5% (v/v) and 1 ml of different concentrations of essential oil (1000 to10 μg/ml with a range of 10 μg/ml) diluted in DMSO).
Bacterial strains were cultured overnight at 37°C in Mueller Hinton Agar (MHA).Test strains were suspended in MHB to give a final density of 10 5 CFU/ml.The mixture (various dilutions of the essential oil + MHB + Tween 80) was placed in Petri dishes, after solidification bacterial strains are inoculated (1 μl containing 10 5 CFU/ml) and the negative control was set up with it.
The MIC is defined as the lowest concentration of the essential oil at which the microorganism tested, does not demonstrate visible growth in the broth after 24 h of incubation at 37°C (Bassole et al., 2003).The MBC is the lowest concentration of essential oil inhibiting any growth visible to the naked eye after 5 days of incubation at 37°C (Mayachiew and Devahastin, 2008).The tests were performed in duplicate and were repeated twice.

Statistical analysis
The obtained yield, antioxidant and antibacterial results were stated in mean ± standard deviation.Significant differences means were determined by Student t-test.Probability values lower than 5% were regarded as significant.

Chemical composition of the essential oil
The oil extracted (2.04 ± 0.34 %) is yellowish in color and has an aromatic odor characteristic of lemon.The yield cited by Himed and Barkat (2014) of the Eureka variety, extracted by cold pressure is 1.02 ± 0.04%.The two yields represent a significant difference; this means that the method of extraction affects the yield.
Regarding the chemical composition of the essential oil tested, this was shown to be a complex mixture of many components.Table 1 show the identified 30 compounds which accounted for 97.8% of the total oil.The essential oil was dominated by limonene (61.3%) followed by βpinene (9.7%), α-citral (4.2%) and α-terpinene (3.8%).Limonene concentration is an essential oil which may vary between 32 and 98%, and that depend on: 32 to 45% in bergamot, 45 to 76% in lemon and 68 to 98% in sweet orange (Moufida and Marzouk, 2003).

Antioxidant property
As shown in the Table 2, the free radical scavenging activity measured by DPPH assay of the essential oil tested is inferior to α-tocopherol with IC 50 (1.01± 0.420 and 0.78 ± 0.130 mg/ml respectively).Oxidation of linoleic acid, in β-carotene/linoleic acid system, is effectively inhibited by the essential oil of C. limon with a value close to α-tocopherol (72.2 ± 0.014% and 72.8 ± 0.079% respectively).No significant differences were reported, it means that C. limon essential oil has significant antioxidant property close to that of αtocopherol.Wei and Shibamoto (2007) showed the presence of a significant antioxidant potential of essential oils rich in hydrocarbon monoterpenes (limonene and β-pinene).Ruberto and Baratta (2000) reported that γ-terpinene could also be taken into account for the antioxidative activity observed, which is for 3.8% oil studied.This activity can also be attributed to the presence of oxygenated sesquiterpenes (Cherrat et al., 2014).Aoyama et al. (1988) reported that terpenes showed a synergistic effect in antioxidation with other antioxidants.Therefore, by inference the higher antioxidant activity in the essential oils might be due to their higher contents of terpenes (with basic structure of isoprene) (Mau et al., 2003).The aldehyde monoterpenes (citral) and hydrocarbon sesquiterpenes (trans-caryophyllene) were responsible for DPPH neutralization (Mimica-Dukic et al., 2004).

Antimicrobial property
The disc diameters of zone of inhibition (DIs), minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of C. limon essential oil for the bacteria tested are shown in Table 3.The correlation between the two different screening methods was examined, and generally have larger zones of inhibition correlated with lower MIC and MBC values.
The essential oil of C. limon showed inhibition zones against all microorganisms tested.This was confirmed by both MICs and MBCs data, where the essential oil exhibited significant antibacterial activity against the microorganisms tested, particularly against gram-positive bacteria (S. aureus and B. cereus) which have the lowest MIC (240 and 300 μg.ml -1 respectively).As cited by Burt (2004) and Hussain et al. (2010), the test Gram-positive bacteria were found to be more susceptible to antimicrobial agents than Gram-negative bacteria.The weaker antimicrobial activity against Gram-negative compared to Gram-positive bacteria is ascribed to the structure of their cellular walls mainly with regard to the presence of lipoproteins and lipopolysaccharides in Gram-negative bacteria that form a barrier to hydrophobic compounds (Inouye et al., 2001).It is well known that the composition, structure, as well as functional groups of
Limonene was present at a very high concentration in the Citrus essential oil.According to Espina et al. (2011), the greater antimicrobial activity of essential oil might not be attributed to limonene, but should be related to the presence of other essential oil constituents; unlike Ruiz and Flotats (2014) who reported that, the documented antimicrobial effect of Citrus essential oil can be attributed to the essential oil or limonene as well, as its main component.
The strong antimicrobial activity of the essential oil against the susceptible microorganisms can be attributed to the presence of high concentration of monoterpenes (Reza et al., 2014), where 78.1% are monoterpenes in the essential oil of C. limon tested.Moreover, oxygenated monoterpenes might be involved in higher antimicrobial activity of studied essential oil.Some authors (Carson and Riley, 1995;Burt, 2004) have demonstrated that oxygenated monoterpenes had an important antimicrobial activity.Nevertheless, the antimicrobial activity of essential oil might also be due to the synergistic interaction of other constituents present in smaller amounts.

Conclusion
In conclusion, essential oil of C. limon (variety Eureka) is a rich source of antioxidant which can be used as powerful herbal antioxidant and the antibacterial property can be considered as an additional health promoting factor.Antioxidant and antibacterial properties are directly related to its chemical composition which is rich in monoterpenes.
Finally, this essential oil could play a beneficial role as a natural preservative ingredient in food and pharmaceutical industries.

Table 1 .
Chemical composition of Citrus limon (Eureka variety) essential oil obtained by hydrodistillation.

Table 2 .
Effects of Citrus limon essential oil and positive control (α-tocopherol) on DPPH and βcarotene/linoleic acid systems.