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Phytochemical Analysis by Gas Chromatography Coupled to Mass Spectrometry (GC-MS)

This is a bio-guided study investigating the antifungal susceptibility of Candida spp. to the action of C. sativum EO and its active fraction. The MIC test was carried out using 96-well U-bottom tissue culture microplates containing 100 ml/well of RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA). A stock solution of EO emulsion and active fraction was transferred to the first well and serially diluted to obtain concentrations ranging from 1,000 to 0.48 mg/ml. Nystatin (Sigma-Aldrich, St. Louis, MO, USA) and Amphotericin B (Unia˜o Quı´mica Farmaceˆutica Nacional, SP, Brazil), and 6.25% propylene glycol (vehicle, v/v) were used as positive and negative controls, respectively. Sterility of the culture medium and the EO emulsion, and yeast viability were also checked. Fungal inocula were prepared (530 nm, abs 0.08–0.1) and diluted to reach a final concentration of 2.56103 CFU/ml in the wells. Plates were incubated at 35uC for 24–48 hrs. MIC was defined as the lowest concentration of the oil that inhibited visible fungal growth.

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