The and antibacterial is established to use them

The present study evaluates the chemical composition,
antioxidant and antimicrobial activities of essential oil of Citrus lemon
(Eureka) extracted by hydrodistillation. The composition of this oil was analyzed
by GC/MS and 27 constituents, which accounted for 92.99 % of the oil, were
identified. The main components were Limonene (61.26%) followed by ?-Pinene
(9.65%), ?-Trpinene (8.16%) and Citral (2.17%). Antioxidant activity of the Citrus
limon essential oil was evaluated by using DPPH radical scavenging and
?-carotene-linoleic acid bleaching. In both tests, the oil showed antioxidant
activity close to the positive control (?-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 micro dilution methods. Citrus
limon essential oil showed antimicrobial effect against all microorganisms
tested.

Keywords: Citrus limon ; Essential
oil ; Hydrodistillation; Antioxidant activity ; Antibacterial
activity.

1.   
Introduction

Essential oils have also diverse and relevant
biological activities. For instance, they are used in the medical field thanks
to their biological activities (bacteriocidal, virucidal and fungicidal) and
medicinal properties (Mayaud et al., 2008). The use of essential oils as
food preservatives has been described by (Burt, 2004 ; Tiwari et al.,
2009). Because of their complex chemical composition, often composed of more
than 100 different terpinic compounds, essential oils have a large biological
and antimicrobial activity spectrum (antibacterial, antifungal, antimoulds,
antiviral, pest control, insect repellents).

Citrus
is the most abundant crop in the world, with about 13 million tons of lemon
products produced during 2004 (Laufenberg et al., 2003). The amount of
residue obtained from Citrus fruit account for 50% of the original
amount of the whole fruit (Chon and Chon, 1997).

Recently, the essential oils and various extracts of
plants have been of great interest since they have been important sources of natural
products. The prolongation of the storage stability of foods, synthetic
antioxidants and antibacterial is established to  use them in industrial process. Although the
side effects of some synthetic conservatives used in food processing have
already been documented (Ames, 1983, Baardseth, 1989). For this reason,
governmental authorities and consumers are concerned about the safety of the
food and also about the potential effects of synthetic additives on health
(Reische et al., 1998),  the
essential oils, despite their wide uses and fragrances, constitute an effective
alternative to synthetic compounds produced by chemical industry without having
any side effects (Faixova et al., 2008).

Our objective in the present study is the extraction
of the essential oil of Citrus limon (Eureka variety) by
hydrodistillation and the evaluation of its antioxidant and antibacterial
activities.

2. Materials and Methods

2.1. Essential oil

2.1.1. Process of extraction

The essential oil of Citrus limon (Eureka)
peels is extracted by steam distillation for 2h and 30 min using a
Clevenger-type apparatus. The supernatant was separated by decantation. The
essential oil was collected, dried over anhydrous sodium sulfate and stored in
glass vials covered by aluminum foil at 4°C until used. The yields were calculated
according to the weight of the plant material before distillation (expressed in
percent, w/w of the fresh vegetable material).

2.1.2. Gaz chromatography-mass spectrometry

The essential oils were analyzed by gas chromatography
coupled to mass spectrometry (GC/MS) using an apolar column (HP5 MS) (30m×0.25mm,
0.25µm film thickness). The Oven temperature was at 60°C and was held for 8min,
then 2°C
to 250°C
for 10min. Helium gas was used as the carrier gas at a constant flow rate of
3ml/min. Injector and MS transfer line temperatures were set at 250°C and 280°C respectively. The
temperature of electronic impact at 70eV source was 230°C. Samples (1µl) were
injected at 250°C
and the split ratio 1:20. Identification of the components was made by
determination of their retention indices (KI) relative to those of a homologous
series of n-alkanes (C8-C28) (Fluka, Buchs/sg,
Switzerland) and by matching their recorded mass spectra with those stored in
the spectrometer database (NIST MS Library v. 2.0) and the bibliography (Adams,
2001). Component relative percentages were calculated based on GC peak areas.

2.1.3. Evaluation of the antioxidant and antiradical
activities

2.1.3.1. Scavenger effect on DPPH

The DPPH
solution was prepared according to the protocol described by Mansouri et al.
(2005), by the solubilization of 2.4 mg of DPPH in 100 ml of methanol. 25 ?l of
each of the methanolic solutions of the essential oils tested or reference
antioxidant (?-tocopherol) wer added to 975 ?l of the methanolic solution of
DPPH. The mixture was left in the dark for 30 minutes and the discoloration
with respect to the negative control containing only the DPPH solution was
measured at 517 nm. The antiradical activity was estimated according to the
following equation (Sanchez-Moreno, 2002):

Antiradicalaire
activity (%) = (A0- At)/A0 × 100

Where At is
the absorbance value of the tested sample and A0 is the absorbance
value of the blank sample. The percentage of inhibition was plotted after 30
min against concentration, and the equation for the line was used to obtain the
EC50 value.

2.1.3.2. ?-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 2mg ?-carotene and 25µl linoleic acid was prepared in 10ml
of chloroform and 200mg Tween 40. The
chloroform was then completely evaporated at 40°C under vacuum. 50ml of
oxygenated distilled water was subsequently added to the residue and mixed
gently to form a yellowish emulsion. The essential oil and ?-tocopherol (positive
control) were individually dissolved in methanol (2mg/ml) and 350 µl volumes of
each of them were added to 5ml of the above emulsion in test tubes and were mixed.
The test tubes were incubated in a boiler at 50°C for 2h together with a
negative control (blank) contained the same volume of methanol instead of the
essential oil. The absorbance values were measured at 470nm on an ultraviolet
and visible (UV-Vis) spectrometer. The antioxidant activities (inhibitions
percentage, I%) of the samples were calculated using the following
equation :

I% = (A?-carotene after 2h assay/Ainitial ?-carotene)
× 100

Where A?-carotene after 2h assay is the
absorbance values of ?-carotene after 2h assay remaining in the samples and Ainitial
?-carotene is the absorbance value of ?-carotene at the beginning of the
experiments.

2.2. Antimicrobial activity

2.2.1. Bacterial strains

The essential oil was tested against 9 strains of food
borne pathogenic bacteria: two Gram positive : Bacillus cereus,
Staphylococcus aureus ATCC 29213 and seven Gram negative : Escherichia
coliATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica,
Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marcescens, Proteus
mirabilis.

Bacterial strains were cultured overnight at 37°C on Mueller Hinton broth
and adjusted to a final density of 106CFU/ml, and used as an
inoculum.

2.2.2. Diffusion assay

In vitro antibacterial activity of the essential oil
evaluated against the 9 bacterial strains by the disk diffusion method (Rota et
al., 2004). The test was performed in sterile Petri dishes containing solid
and sterile Mueller-Hinton agar medium. The essential oil (5µl) absorbed on
sterile paper discs (Whatman disc of 6mm diameter), were placed on the surface
of the media previously inoculated with 100 µl of microbial suspension (106
CFU/ml) then the Petri dishes were incubated at 37°C for 24h after staying at 4°C for 2h. The inhibition zone
diameters around each of the disks (diameter of inhibition zone including the
disc diameter) were measured in millimeters.

2.2.3. Determination of minimal inhibitory
concentration (MIC)

The minimal
inhibition concentration (MIC) values were studied for the bacterial strains
which were sensitive to the essential oil in disc diffusion assay. Minimal
inhibition concentration (MIC) values were determined by broth micro dilution
method (Carson et al., 1995). The test was performed in Mueller Hinton
broth (MHB) supplemented with Tween 80 (concentration of 0.5% (v/v) and 1ml of different
concentrations of essential oil (1000- 10?/ml with a range of 10 ?g/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 105 CFU/ml.
The mixture (various dilutions of the essential oil + MHB + Tween 80) is placed
in Petri dishes and
after solidification, the bacterial strains were inoculated (1?l containing 105
CFU/ml), the negative control was set up with. The MIC is defined as the lowest
concentration of the essential oil at which the microorganism tested does not show any visible growth in the broth after 24h of
incubation at 37°C
(Bassolé et al., 2002). 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 &
Devahastin, 2008). The tests were performed in duplicate and repeated twice   

2.3. Statistical analysis

The obtained yield, antioxidant and antibacterial
results were stated in mean ± standard deviation. Significant differences
between means were determined by Student test. P values inferior to 0.05 were
regarded as significant.

3. Results and discussion

3.1. Chemical composition of the essential oil

2.38 ± 0.08% oil
extracted are yellowish and have 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 (p<0.05); this means that the method of extraction affects the yield. Regarding the chemical composition of the essential oil tested, it was shown to be complex mixtures of many components. Table 1 shows the identified compounds (27 compounds) which accounted for 92.99% of the total oil, modes of identification, and percentage obtained by GC/MS, as well as the retention indices (I) listed in order of their elution from the  HP5 MS capillary column. The essential oil was dominated by Limonene (61.26%) followed by ?-Pinene (9.65%), ?-Terpinene (8.16%) and Citral (2.17%). The main component is limonene. Its concentration in the essential oil is varied between 32% and 98%, depending on the variety: 32-45% in bergamot, 45-76% in lemon and 68-98% in sweet orange (Moufida and Marzouk, 2003). 3.2. Antioxidant activity The essential oil was a subject to screening for its possible antioxidant activity by two complementary test systems namely DPPH free radical scavenging and ?-carotene/linoleic acid systems. As shown in the table 2 the free radical scavenging activity measured by DPPH assay of the essential oil tested is inferior to ?-tocopherol with IC50 (0.60 ± 0.04mg/ml and 0.81 ± 0.03 mg/ml respectively). In ?-carotene/linoleic acid system, oxidation of linoleic acid is effectively inhibited by the essential oil of Citrus limon with a value close to that ?-tocopherol (0.53 ± 0.01mg/ml and 0.84 ± 0.03mg/ml respectively). according to these results, the essential oil has shown antioxidant activity comparable to that of ?-tocopherol. Antioxidant activity of an essential oil is attributed mainly to its major components, although the synergistic or antagonistic effect of one compound in minor percentage of the mixture has to be considered (Burt, 2004). Wei and Shibamoto (2007) showed the presence of a significant antioxidant potential of essential oils rich in hydrocarbon monoterpenes (limonene and ?-pinene). Ruberto and Baratta (2000) reported that ?-terpinene could also be taken for its antioxidative activity, which is for 3.84 % oil studied. This activity can be also attributed to the presence of oxygenated sesquiterpenes (Cherrat et al., 2014). Aoyama et al. (1988) reported that terpenes showed a synergistic effect in ant oxidation with other antioxidants. Therefore, the higher antioxidant activity in the essential oils might be due to their higher continence of terpenes (with basic structure of isoprene) (Mau et al., 2003). The aldehyde monoterpenes (citral) and hydrocarbon sesquiterpenes (trans-caryophyllene) were responsible of the DPPH neutralization (Mimica-Dukic et al., 2004). 3.3. Antimicrobial activity The disc diameters of zone of inhibition (DIs), minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of Citrus limon essential oil for the bacteria tested are shown in table 3. The correlation between the two different screening methods was examined and generally larger zones of inhibition correlated with lower MIC and MBC values. The essential oil of Citrus 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 (Staphylococcus aureus and Bacillus cereus) which have the lowest MIC (240 and 300?g.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 lower antimicrobial activity against Gram-negative compared to Gram-positive bacteria is attributed to the structure of their cellular walls mainly in 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 essential oil play an important role in determining their antimicrobial activity. It has been demonstrated that the essential oils exercise their antimicrobial activity by causing structural and functional damages to the microbial cell membrane (Goni et al., 2009). Limonene was present at 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 it should be related to the presence of other essential oil constituents. Ruiz and Flotats (2014) reported that the documented antimicrobial effect of Citrus essential oil can be found attributed to the essential oil or to limonene as well, as its main component. The strong antimicrobial activity of the essential oil against almost all the susceptible microorganisms can be attributed to the presence of high concentration of monoterpenes (Reza et al., 2014), where 78.13% are monoterpenes in the oil tested. Moreover, oxygenated monoterpenes might be involved in the higher antimicrobial activity of studied essential oil. Some authors (Burt, 2004; Carson et Riley, 1995) 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. 4. Conclusion In conclusion, essential oil of Citrus limon (variety Eureka) is a rich source of antioxidant and can be used as powerful herbal antioxidant. Its antibacterial property could 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.

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