Chemical Composition and Biological Potential of Essential Oil from ...

a mass selective detector (MSD5975B, ionization voltage 70eV; all Agilent, Santa Clara, CA). The carrier gas was He andwas used at 1 mL.min -1 flow rate. The oven temperatureprogram was as follows: 1 min at 100°C ramped from 100 to260°C at 4°C.min -1 and 10 min at 260°C. Thechromatograph was equipped with a split/splitless injector usedin the split mode. The split ratio was 1:100. Identificationof components was assigned by matching their mass spectrawith Wiley and NIST library data, standards of the maincomponents and comparing their Kovats retention indices withreference libraries (Bouaziz et al., 2009) and from the literature.The component concentration was obtained bysemi-quantification by peak area integration from GC peaksand by applying the correction factors.2.4. Antioxidant Potential of Essential oil2.4.1. DPPH assayDPPH assay was carried out as described by Bouaziz et al(2010). Various concentrations of essential oil in ethanol (50µL) were mixed with methanolic solution containing DPPHradicals (6 µM). The mixtures were shaken vigorously andleft to stand for 30 min in the dark. Decrease in colorizationwas measured spectrophotometrically at 517 nm using aUV-1800PC, Japan spectrophotometer. The radicalscavenging activity (RSA) was calculated using the equation:% RSA = [(A DPPH – A E ) / A DPPH ] × 100Where A E was the absorbance of solution containingantioxidant extract and A DPPH was the absorbance of DPPHsolution. The extract concentration providing 50% inhibition(IC 50 µg.mL -1 ) was calculated from the graph of RSApercentage against extract concentration. 2, 6-di-tert-butyl-4-hydroxy-boxylic acid (BHT) was used as referencecompound.2.4.2. ABTS assayThe free-radical scavenging capacity was measured usingthe ABTS decoloration method (Re et al., 1999) with somemodifications. Briefly, ABTS was dissolved in water to get a7 mM concentration. ABTS radical (ABTS .+ ) was producedby reacting this stock solution with a 2.45 mM K 2 S 2 O 8 solutionand allowing the mixture to stand in the dark at roomtemperature for 12-16 hours before use. The ABTS .+ solutionwas diluted with ethanol to an absorbance of 0.70 ± 0.02 at730 nm. After the addition of 100 µL of sample, ethanol as ablank, or Trolox standard to 2.9 mL of diluted ABTS .+ solution,absorbance readings were taken after 6 min. ethanolic solutionsof known concentrations of Trolox were used forcalibration. This standard curve was linear between 0.2 mMand 0.8 mM of Trolox (y = 0.6792 x, r² = 0.9965). TheResults were expressed in TEAC (mM).2.5. Antimicrobial activity2.5.1. Test organisms and culture mediaThe most of the test organisms used were from theinternational culture collection strains of Gram-positivebacteria Staphylococcus aureus ATCC 6538, Enterococcushirae ATCC 10541, Bacillus subtilus ATCC 6633,Gram-negative bacteria Pseudomonas aeruginosa ATCC15442, Escherichia coli ATCC 10536, Salmonellatyphimurium TA 97, the yeast mould Candida albicans ATCC10231, and filamentous fungi Aspergillus niger ATCC 16404,Trichoderma reesei Rut C30.The bacteria were cultivated in Tryptic Soy Broth (TSB) orAgar (TSA) (Sigma) at the appropriated temperature (30°C or37°C) of the strain. Fungi and yeasts were cultured on MaltExtract Broth (MEB) or Agar (MEA) (Fluka) at 28°C.Inocula were prepared by adjusting the turbidity of eachbacterial and yeast cultures to reach an optical comparison tothat of a 0.5 McFarland standard, corresponding toapproximately 1-5 10 8 cfu.mL -1 . The concentration of sporesuspensions was determined using a hematocytometer (Thomacell) and adjusted to 1-5 10 8 spores mL -1 .2.5.1. Minimal Inhibitory Concentration (MIC), MinimalCidal Concentration (MCC)This test was used to determine MIC and MCC of theessential oil of C. sempervirens against the test organisms asrecommended by the National Committee for ClinicalLaboratory Standard (NCCLS, 2002). This test wasperformed in a sterile 96-well microplate. The inhibitoryactivity of the essential oil was properly prepared andtransferred to each microplate well in order to obtain a twofoldserial dilution of the original sample. To obtain stablediffusion, a stock solution of the essential oil was prepared in0.1% ethanol. The inocula (100 µL) containing 104 cfu ofbacteria were added to each well. Control wells containedbacteria or fungi only, adequate medium only andchloramphenicol or gentamycin antibiotics (for bacteria andfungi respectively) (10 µg.L -1 ). Thereafter, 30 µL of 0.02%resazurin and 12.5 µL of 20% Tween 80 were added. Plateswere aerobically incubated at 30°C for 16-20 h. Afterincubation, the wells were observed for a color change fromblue to pink. MIC was defined as the lowest concentration atwhich no growth was observed (blue colored) after incubation.Ethanol at 0.1% had no inhibition effect.To determine MCC values, 10 µL of each culture mediumwith no visible growth were removed of each well andinoculated in TSA or MEB plates. After aerobic incubation at30°C during 16-20 h and the number of surviving organismswas determined.