Evaluation of Antioxidant and Antimicrobial Properties of the Angolan Cymbopogon Citratus Essential Oil With a View to Its Utilization as Food Biopreservative

It was studied the chemical composition, antioxidant, antibacterial and antifungal properties of the essential oil obtained from the Cymbopogon citratus of Angolan origin. Its major constituents analyzed by GC-MS were α-citral (40.55%), β-citral (28.26%), myrcene (10.50%) and geraniol (3.37%). The essential oil antioxidant capacity was statistically identical to that of synthetic antioxidants (DPPH IC50 of 41.7 μg/ml) and superior to that of extracts obtained from fresh leaves of the plant (DPPH IC50 of 55.7 μg/ml). The oil also demonstrated to possess high antibacterial activity even against multidrug resistant strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Klebsiella pneumoniae and antifungal activity against Candida albicans and Non-Candida albicans, Candida parapsilosis and Candida tropicalis. The results of this study, in conjunction with already published data on the properties of other Cymbopogon citratus oils, provide evidence that it could have a potential application as food preservative.

not change the organoleptic characteristics of the food.Interesting results have already been reported with EOs from various plants (Hyldgaard et al., 2012;Sessou et al., 2012).This paper describes the research done on the EO of Cymbopogon citratus (lemongrass) of Angolan origin.The EO of C. citratus is already in use by the pharmaceutical industry as a source of new phytochemical molecules for the development of new drugs.Previous studies on the leaf EO of C. citratus revealed, antityrosinase and antioxidant activities in human cells (Saeio et al., 2011), anti-inflammatory in rats (Gebnou et al., 2013), anti-carcinogenic effects and cholesterol reduction in mice (Costa et al., 2011;Bidinotto et al., 2012).Furthermore, many studies have reported the antimicrobial activity of lemongrass oil originating from different parts of the globe against a diverse range of microorganisms comprising Gram positive and Gram negative bacteria, yeast and fungi namely Bacillus cereus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae (Naik et al., 2010), Salmonella choleraesuis, Pseudomonas aeruginosa, Staphylococcus aureus (Falcão et al., 2012) and Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Alternaria alternata, Penicillium citrinum, Curvularia lunata and Trichoderma harzianum (Mahanta et al., 2007).In the work referred to in this article the objective was to evaluate the antimicrobial activity of the cymbopogon citratus on multidrug resistant strains of Staphylococcus (aureus and epidermidis) Escherichia coli, Klebsiella pnemoniae and fungi (Candida albicans, tropicalis and parapsilosis).All these microorganisms may be food-borne and by this route represent a severe public health problem in large scale (Easa, 2010).Food-borne diseases caused by bacteria and yeast are of major concern worldwide even in developed countries were up to 30% of the population have been reported to suffer from them every year.The severity of the problem might become more pronounced due to the proliferation of antibiotic-resistant strains (McAdam et al., 2012).Among the predominant bacteria involved in food-borne diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of a food in which enterotoxigenic staphylococci have grown and produced toxins.These are considered a potential biological threat because of their stability at high temperatures (100°C for 1 h).While not regarded as highly lethal agents due to the low mortality associated with the illness, staphylococcal enterotoxins can incapacitate individuals for two weeks (Schelin et al., 2011).Moreover it is predictable that S. aureus will continue to develop new virulence characteristics and new patterns of synthetic antibiotic resistance (Fry et al., 2013).A similar drug resistance phenomenon is supposedly occurring with S. epidermidis (Widerström et al., 2012).E. coli is reported to cause bloody diarrhea and can sometimes cause kidney failure and even death.K. pneumoniae is second only to E. coli as a urinary tract pathogen and cases of enteroinvasive Klebsiella pneumoniae and Escherichia coli sepsis from contaminated hamburgers are documented (Sabota et al., 1998;Kim et al., 2005).As in the case of bacteria previously mentioned, Klebsiella infections are encountered far more often now than in the past probably due to the emergence of increasingly resistant strains (Saha et al., 2008).Apart from bacteria, fungal contamination of food is practically inevitable.Candida as many other yeasts and molds is undesirable in many foods manly due to its food spoiling activity, nevertheless, dangerous levels of candida have been found in yoghurt (Montagna et al., 1998).Candida spp. is responsible for the most prevalent opportunistic fungal infections in humans and of particular significance in immunocompromised individuals (Muñoz et al., 2006;Rodloff et al., 2011).
Additionally, and as this is an important characteristic for a food preservative, the antioxidant capacity of the Angolan Cymbopogon citratus EO was evaluated and compared with that of synthetic antioxidants.The use of natural substances to replace the synthetic antioxidants in food industry can result in important health benefits, since the latter have been associated with genotoxic and carcinogenic effects (Kahl et al., 1993, Ndhlala et al., 2010).

Plant Source
The fresh aerial parts of C. citratus were obtained from a local market in Benguela, (latitude 12º58′ south; longitude 13º408′ east), Angola.The plants were identified by a taxonomist (Dr.Pedro Catarino Pires, ISPB, Benguela) and deposited in the Instituto Superior of Saúde de Benguela, ISPB, Benguela.The fresh plant sample was submitted to distillation (4 h) using a Clevenger apparatus to obtain the EO for further analysis.The procedure followed was that described in the European Pharmacopoeia (2011).The yield in EO was of 1.3%, corresponding, on average, to 1.5 ± 0.4 ml of EO per 111 g of fresh plant sample.A total of six samples of fresh plant, purchased in different days were used to obtain the six samples of EO.

Isolation, Characterization and Identification of Constituents
The EO was analyzed by GC-MS carried out in a Hewlett-Packard 6890 gas chromatograph fitted with a HP1 fused silica column, interfaced with an Hewlett-Packard mass selective detector 5973 (Agilent Technologies) operated by HP Enhanced Chem-Station software, version A.03.00.Components were identified by their retention indices on both SPB-1 and SupelcoWax-10 columns and from their mass spectra.Retention indices, calculated by linear interpolation relative to retention times of C8-C23 of n-alkanes, were compared with those of authentic standards included in our own laboratory database.

Total Phenolic Contents
The amount of total phenolic compounds was determined in the aerial parts of C. citratus according to Jang et al. (2007) using the Folin-Ciocalteu reagent.Each sample was extracted using methanol, for one hour.In brief, 2.5 g of a dried leaves sample was added to 100 ml of the solvent, under constant agitation for 1 hour.Afterwards, the solid was separated from the extract through vacuum filtration and a volume of 0.5 ml of each extract was added to 0.5 ml of Folin-Ciocalteu reagent.The mixture was left to rest for 3 min at room temperature before 1.0 ml of sodium carbonate 6% was added to it.After standing at room temperature for 120 min, absorbance readings were performed at 720 nm using a UV-Vis spectrophotometer (Beckman DU-64 spectrophotometer, Beckman Instruments Inc., Fullerton, CA).Total phenolics were quantified by means of a calibration curve obtained from measuring the absorbance of gallic acid standards.Results were expressed as milligrams of gallic acid equivalents per 100 g of fresh sample weight.

Antioxidant Activity
The antioxidant activity of C. citratus EO was evaluated by measuring the bleaching of the purple-colored ethanolic solution of 2,2-diphenyl-1-picrylhydrazyl free radical (DPPH • ).This methodology was performed using an ELX80 Microplate Reader (Bio-Tek) in the following way: the reaction mixture in each one of the 96-wells consisted of one of the different concentration of EO (20 μL) and 280 μL methanolic solution of DPPH radical (0.5 mM).After 30 minutes incubation, the changes in color (from deep-violet to light-yellow) were measured at 517 nm (Shimadzu, model UV-1800), according to the method previously reported (Brand-Williams et al., 1995).The butyl hydroxyl toluene (BHT) was used as a positive control while ethanol was the negative one.
The percentage of inhibition ( % I ) of DPPH radical was computed through the equation: where, A s represents the absorbance of the solution of the sample extracts containing DPPH • , and A DPPH is the absorbance of the DPPH • solution.All determinations were performed in triplicate.The concentration of extract sample required for 50% inhibition of the DPPH free radical (IC 50 ) was also calculated.

Microorganisms
The

Propagation and Maintenance of Microorganisms
The studied microorganisms were streaked on the nutrient agar slants and then incubated overnight at (37±1 ºC).The cultures were kept under refrigerated conditions and were sub-cultured every fifteen days.

In Vitro Testing
Empty sterilized discs were impregnated with 5µl of oil diluted with DMSO to obtain different concentrations (5%, 10%, 20%, 40%, 60%, 80% and 100%).DMSO was used as negative control.The inoculated plates were incubated at 37ºC for 24 or 48 hours for bacterial and fungal assessment, respectively.Antibacterial activity was evaluated by measuring the zone of inhibition in millimeters after the incubation period.All experiments were done in triplicate and the results are presented as mean±standard deviation of three independent experiments.

In Vitro Antifungal Activity
Antifungal activity was also evaluated by the disk diffusion method in accordance to CLSI guidelines M44-A2.Candida spp.were tested in agar Miller Hinton (DIFCO) supplemented with 2% of glucose and 0.5 g/ml of methylene blue.Yeast suspension was adjusted to a final concentration between 1 and 5 x 10 6 cells/ml and incubated at 35ºC during 48 hours.The antifungal fluconazole, at a concentration of 5µg/disc, was the positive control to Candida spp.Results were interpreted according to CLSI guidelines (2012).

Statistical Analysis
Data are reported as mean±standard deviation of nine measurements.Statistical analysis was performed using the statistical software package SPSS v 20.0 (SPSS for Windows; SPSS Inc., Chicago, IL).Mean comparison was made through an independent sample t-test.Levene's test was utilized to assess the equality of variances.One-way ANOVA was used to compare three or more groups, and post-hoc Dunnett's test was performed for simultaneous paired comparisons.P values less than 0.05 (95% confidence level) are reported as "statistically significant".

Results and Discussion
The results of the chromatographic analysis of the C. citratus EO presented in Table 1 reveal that the chemical compositon of C. Citratus EO obtained from plants grown in Angola is similar to that of plants from other provenances (Sessou et al., 2012;Tajidin et al., 2012;Masamba et al., 2003).Accordingly, the major constituents are α-citral (40.55%), β-citral (28.26%), myrcene (10.50%) and geraniol (3.37%).The volatile fraction of C. citratus EO revealed the presence of 10 different compounds accounting for 83.86% of total peak area.The preservation of food generally requires the use of various compounds to perform a range of functions namely antioxidants, antibacterial and antifungals.The ideal would be to find a single biopreservative that could perform all these functions simultaneously.In order to assess the antioxidant potential of the C. citratus EO, its free radical scavenging activity by the DPPH assay was measured (Figure 1).

Table 1 .
Angolan C. citratus EO main components as revealed by GC-MS