American Journal of Plant Sciences, 2014, 5, 299-305 Published Online February 2014 (http://www.scirp.org/journal/ajps) http://dx.doi.org/10.4236/ajps.2014.53041 Use of Essential Oils of the Genus Citrus as Biocidal Agents Marcos S. Gomes1, Maria das G. Cardoso1*, Maurilio J. Soares2, Luís R. Batista3, Samísia M. F. Machado4, Milene A. Andrade1, Camila M. O. de Azeredo2, Juliana Maria Valério Resende3, Leonardo M. A. Rodrigues3 1Department of Chemistry, Federal University of Lavras, Lavras, Brazil; 2Laboratory of Cell Biology, Instituto Carlos Cha- gas/Fiocruz, Curitiba, Brasil; 3Department of Food Science, Federal University of Lavras, Lavras, Brazil; 4Department of Chemistry, Federal University of Sergipe, São Cristóvão, Brazil. Email: *[email protected] Received November 26th, 2013; revised December 28th, 2013; accepted January 18th, 2014 Copyright © 2014 Marcos S. Gomes et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor- dance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of the intellectual property Marcos S. Gomes et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian. ABSTRACT In this study, the essential oils extracted from the peels of Citrus aurantifolia, Citrus limon and Citrus sinensis were chemically characterized and quantified. These essential oils and their standards limonene, citral and li- monene + citral were evaluated (at concentrations ranging from 500 to 3.91 mL·mL−1) regarding their anti-try- panosome, antifungal and antibacterial activities. The chemical identification and quantification (by GC/FID and GC/MS) displayed the limonene as the major component of the three essential oils. It did not verified anti- trypanosome activity for all treatments. Regarding antimicrobial activity, the results were satisfactory against Gram-negative bacteria (Staphylococcus aureus, Listeria. monocytogenes and Salmonella cholerasuis) and funghi (Aspergillus niger, A. flavus and A. carbonarius), except for the bacterium Pseudomonas aeruginosa, which showed no inhibition for any treatment used. KEYWORDS Volatile Essential Oil; Trypanosoma cruzi; Gram-Negative Bactéria; Funghi; Antimicrobial 1. Introduction bacterial properties [4-7]. It has been reported that the antimicrobial properties of essential oils are due to hy- Human civilization has been using plants with thera- drophobic characteristic of the chemical constituents of peutic properties for centuries and these natural products their composition [8]. The lipophilicity of the essential nowadays have been used in the development of new oil allows interaction among their constituents with lipids drugs in the Pharmaceutical area. Among the compounds in the cell membrane, affecting the cell permeability and used to produce these drugs, the secondary metabolites causing changes in cell structure [9]. have been raised as one of the most important molecules. Despite recent advances in antimicrobial chemother- In this context, we highlight the essential oils, which are apy, the treatment of parasitic diseases has remained as a mixtures of several volatile compounds being insoluble public health problem highlighting the Chagas disease in water, soluble in organic solvents and showing great [10]. This disease is caused by the protozoan parasite therapeutic importance [1]. Trypanosoma cruzi, causing approximately 50,000 deaths In recent decades, the antimicrobials of traditional per year [11]. In recent years, the use of essential oils has plants have received great attention due to the resistance acquired great importance for the treatment of parasitic to commercial antibiotics developed by some microor- diseases [12,13]. ganism [2,3]. An alternative to reduce the microorganism The development of new effective drugs against para- growth is using essential oils with antifungal and anti- sitic disease (i.e. Chagas disease) has been aimed for *Corresponding author. several researchers, because most of the synthetic com- OPEN ACCESS AJPS 300 Use of Essential Oils of the Genus Citrus as Biocidal Agents pounds used to treat these parasitic diseases produce tox- model QP 5050A), equipped with fused silica capillary icity and side effects for the patients. Moreover, there has column (J&W Scientific; 5%-phenyl-95%-dimethyl- been a need to find more effective compounds with anti- polysiloxane) 30 m × 0.25 mm i.d., 0.25 μm of film, us- −1 microbial effect to reduce the contamination and deteri- ing He as gas carrier, with flow of 1.2 mL·min . The oration of food. chromatographic conditions of the analysis were the Thus, in this study we aimed to characterize and che- same used for CG-DIC. The conditions of the EM were mically quantify the essential oils extracted from the detector of ionic capture operating by electronic impact peels of lime (Citrus aurantifolia), limon (Citrus limon) and impact energy of 70 eV; scan rate 1.000; interval of and orange (Citrus sinensis), as well as to evaluate, with decomposition of 0.50 fragments/s and detected frag- standards limonene, citral and limonene + citral, the anti- ments in the stripe from 40 to 500 Da. For the identifica- trypanosome, antifungal and antibacterial activities. tion of the constituents, it was also used the comparison of their retention indexes with the ones of the literature 2. Material and Methods [15]. For the retention index, the equation of Van den Dool and Kratz (1963) was used in relation to a homolo- 2.1. Collect of the Vegetal Material and gous series of n-alkanes (nC9-nC18). Two libraries Extraction of the Essential Oil NIST107 and NIST21 were also used, which allowed The vegetal materials, lime (C. aurantifolia), lemon (C. comparison of the spectra database with the ones in the limon) and orange (C. sinensis), were collected on a rural libraries. area of Lavras city in the morning during the months of March and April 2012, at coordinates 21˚26'27 south and 2.3. Evaluation of the Anti-Trypanosome in 44˚39'27 west, with an altitude of 1049 meters. Vitro Activity The extraction process of the essential oils was per- First, confirm that you have the correct template for your formed at Laboratory of Organic Chemistry of the De- paper size. This template has been tailored for output on partment of Chemistry at Federal University of Lavras. the custom paper size (21 cm × 28.5 cm). Evaluation of The method used was the hydrodistillation, using a mod- the anti-trypanosome activity of the essential oils, citral ified Clevenger device [14]. Extractions were perform- and limonene standards and their mixture (limonene + ed in triplicate for a period of 2 hours and their yields citral) were performed at the Instituto Carlos Chagas/ have been calculated on moisture free basis. Moisture Fiocruz, Curitiba, Paraná. Three-day-old culture epimas- was performed using the technique described by [4], us- tigote forms of Trypanosoma cruzi strain Dm28c were ing the Dean Stark type glass collector. The humidity used in all experiments. The parasites were kept at 28˚C was calculated considering the water content in 100 g of in LIT (liver Infusion Tryptose) medium supplemented sample. with 10% fetal calf serum. The essential oils were initially dissolved in dimethyl 2.2. Chemical Identification and Quantification sulphoxide (DMSO) at a concentration of 100 mg·mL−1. of the Essential Oils This solution was then dissolved in culture medium to −1 The quantitative analysis and the identification of con- obtain a stock solution at 1 mg·mL . Under this condi- stituents of the essential oils were performed at the De- tion, the DMSO was diluted at 0.01%, a concentration partment of Chemistry at Federal University of Sergipe. that is not toxic for the protozoa. Both solutions were The quantitative analyses of the reaction mixture were stored at −20˚C. The stock solution was then diluted at performed in a gas chromatography equipped with a different concentrations for the experiments (500; 250; −1 flame ionization detector (Shimadzu GC-17A), under the 125; 62.5; 31.25; 15.63; 7.81; 3.91 µg·mL ) in 20 μL of following operational conditions: fused silica capillary LIT medium in each well of 96-wells plates. After dilu- column ZB-5MS (5% dimethylpolysiloxane) with 30 m × tion, it was added 180 μL of culture medium with epi- 0.25 mm i.d. × 0.25 μm of film, using He as gas carrier mastigote per well, at a concentration of 1 × 107 parasites with flow of 1.2 mL·min−1. The temperature was kept at mL−1. In control wells were added 200 μL of culture me- 50˚C for 2 minutes, and later increased 4˚C min−1, up to dium with parasites, but without treatment. The plates reaching 200˚C. After that, increased 15˚C min−1, up to were incubated for 24 hours at 28˚C. reaching 300˚C, keeping this temperature steady for 15 After incubation, 50 μL of MTT (3-(4,5-methyl-thia- minutes; the temperature of the injector was 250˚C and zol-2-yl)-2,5-diphenyltetrazolium bromide) solubilized at the temperature of the detector (or interface), 280˚C. It 10 mg·mL−1 in PBS (phosphate buffered saline, pH 7.2) was injected a volume of 0.5 µL of the reaction mixture was added to all wells. The plate was wrapped in alumi- dissolved in ethyl acetate. The qualitative analysis of the num foil and incubated for 3 hours at 28˚C [16]. After reaction mixture was performed in gas chromatography this second incubation, 50 μL of paraformaldehyde (4%) coupled to a mass spectrometry CG-EM (Shimadzu, in PBS pH 7.2 were added and the plate was centrifuged OPEN ACCESS AJPS Use of Essential Oils of the Genus Citrus as Biocidal Agents 301 at 1700 rpm for 10 minutes.