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On-line version ISSN 1678-4324
Braz. arch. biol. technol. vol.55 no.4 Curitiba July/Aug. 2012
ENGINEERING, TECHNOLOGY AND TECHNIQUES
Influence of essential oil fractionation by vacuum distillation on acaricidal activity against the cattle tick
Fernando Cidade TorresI,II; Aline Machado LucasI; Vera Lucia Sardá RibeiroII; João Ricardo MartinsIII; Gilsane von PoserIV; Maria Silvia GualaV; Heriberto Victor ElderV; Eduardo CasselI
ILaboratório de Operações Unitárias; Faculdade de Engenharia; Pontifícia Universidade Católica do Rio Grande do Sul; Av. Ipiranga, 6681; 90619-900; Porto Alegre - RS - Brasil, E-mail: firstname.lastname@example.org
IILaboratório de Acarologia e Entomologia; Faculdade de Veterinária; Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9090; 91540-000; Porto Alegre - RS - Brasil
III Instituto de Pesquisas Veterinárias Desidério Finamor; Estrada do Conde s/n; Guaíba - RS- Brasil
IVPrograma de Pós-Graduação em Ciências Farmacêuticas; Universidade Federal do Rio Grande do Sul, Av. Ipiranga, 2752; 90610-000; Porto Alegre - RS - Brasil
VFacultad de Ingeniería Química; Universidad Nacional del Litoral; Santa Fe - Argentine
The aim of this work was to study the influence of essential oil fractionation on acaricidal activity against the cattle tick Rhipicephalus (Boophilus) microplus. The citronella (Cymbopogon winterianus J.) and pepper tree (Schinus molle L.) essential oils were fractionated by vacuum distillation yielding fractions that were analyzed by the GC/MS. Laboratory tests were carried out to determine the effect of the total essential oil and fractions on larvae of the cattle tick R. (B.) microplus. The fractions 04 and 05 of the C. winterianus essential oil were the most active showing LC50 values of 1.20 and 1.34 μL/mL, respectively. The LC50 of the total oil was 3.30 μL/mL while the effect of the fractions 01, 02 and 03 was less pronounced, with LC50 values of 4.37, 4.24 and 3.49 μL/mL, respectively. The fraction 03 of the S. molle essential oil was the most active showing LC50 value of 8.80 μL/mL while the fractions 01 and 02 did not show toxic effects on the larvae.
Key words: Cymbopogon winterianus J., Schinus molle L., acaricidal activity, vacuum distillation, Rhipicephalus (Boophilus) microplus
The cattle tick Rhipicephalus (Boophilus) microplus represents a major problem for the cattle production in several regions of the world. It causes blood loss, reduction in both weight gain and milk production. It also serves as a vector for certain diseases such as bovine babesiosis, a cattle tick-borne disease caused by the haemoprotozoan parasites of the genus Babesia, and anaplasmosis, caused by obligate intracellular bacteria of the genus Anaplasma that parasitize erythrocytes and monocytes of higher vertebrates, mostly ruminants. In addition, the parasite causes damage to skin of the cattle directly affecting the value of hides for leather manufacture (García-García et al. 1999; Ducornez et al. 2005). The economical losses world-over were estimated as $7 billion (Castro-Janer et al. 2009) due to this.
The most extensively used methods for tick control involve the application of acaricides such as pyrethroids and formamidine, both in the environment and directly on the animal skin. However, the indiscriminate use of these substances leads to increase in acaricide resistance and environmental contamination (Chagas et al. 2002; Chagas 2004). Therefore, effective and easily accepted methods of tick control are extremely necessary (Cordovés 1997).
Natural products can be considered a valuable alternative. A large number of plants and entomopathogenic agents have been reported to have acaricidal effect against the diverse stages of several species of ticks such as Ixodes scapularis and R. (B.) microplus (Borges et al. 2003; Freitas-Ribeiro et al. 2005; Dietrich et al. 2006; Ribeiro et al. 2007; Fernandes et al. 2008; Ribeiro et al. 2008). Among them, essential oils components are known to possess repellent, chemosterilant, antifeeding and biocidal activities against different acarus, showing promissory potential as products for tick control, since some of them are selective and have little or no harmful effects on non-target organisms (Facey et al. 2005; Panella et al. 2005; Dietrich et al. 2006; Tunon et al. 2006; Apel et al. 2009).
The genus Cymbopogon, family Poaceae, comprises a group of about 40 tropical aromatic grasses. The essential oils from several species of this genus have demonstrated insecticidal and repellent activity (Kim et al. 2004; Ketoh et al. 2006; Samarasekera et al. 2006). The citronella (Cymbopogon winterianus J.) essential oil presents repellent properties and represents a significant parcel of the essential oil national production being used in sprays, soaps, candles and other mosquito repellent products (Tanu and Adholeya 2004). Studies performed with the oil distilled from the leaves of citronella showed high acaricidal activity against the larvae and adult females of the cattle ticks when pure oil and diluted (1:4) in ethanol (Chungsamarnyart and Jiwajinda 1992) was used. Olivo et al. (2008) investigated the effect of the essential oil of citronella on R. (B.) microplus engorged females and reported 90% of egg laying inhibition at the concentrations of 1, 10, 25 and 100%. Other study carried out with C. winterianus essential oil demonstrated that the concentrations of 6.1 and 4.1% were lethal to engorged females and larvae of R. (B.) microplus, respectively. Hatching was not observed in the eggs from the engorged females treated with the essential oils at the concentration of 7.14% and egg lying was inhibited by the oil at 10%. Citronellal, geraniol and citronellol were the main components identified in the essential oil (Martins 2006).
Schinus molle L. (Anacardiaceae) is a tree from the South America, originating from Peru (Huerta et al., 2010). It has active substances, such as terpenes, tannins, alkaloids, flavonoids, essential oils, and oleoresins, mainly in the leaves and fruits (Ferrero et al. 2007). Ferrero et al. (2006 2007) studied the insecticidal and repellent effects of the extracts from S. molle. Hayouni et al. (2008). Investigated the antimicrobial activity of pepper tree essential oil.
In this work, the essential oils of C. winterianus and S. molle were fractionated by vacuum distillation (Babu and Kaul 2007) and tested against R. (B.) microplus.
MATERIAL AND METHODS
Plant material and extraction of the essential oil
The aerial parts of C. winterianus were collected from the Experimental Farm of Tekton Óleos Essenciais Ltda in the state of Rio Grande do Sul. The essential oil was extracted from the fresh aerial parts by steam distillation in the Tekton industrial distillery (Cassel and Vargas 2006; Cassel et al. 2009). The average yield of the extraction, 0.62% p/p, was calculated on a wet weight basis from the relationship between the essential oil mass and mass of leaves of the fresh aromatic plants.
The S. molle essential oil was extracted from the fresh plant (fruit 25% p/p; leaves 65% p/p; stems 10 % p/p) by steam distillation. The average yield of the pepper tree essential oil on industrial scale was 0.30% p/p. The fresh plants were collected and the essential oil was extracted in Experimental Center Angel Gallardo.
Essential oil fractionation
The essential oil was fractionated using a vacuum distillation apparatus (Fig. 1). A volume of 300 mL was introduced in a round bottom flask that presented three outputs: output B had a capillary tube aiming to control and stabilize the ebullition inside the round bottom flask; the output TC1 was used to introduce a temperature sensor for the control of essential oil temperature during the distillation in order to avoid very high temperatures, and the third output was employed to connect the packed column.
The fractionating column was 1.5 m in height and was packed with titanium alloy. The packing material was selected in order to protect against the corrosion. The measure of temperature at the top of the fractionating column was determined by the thermocouple with a precision of ± 1.5 °C. Temperatures in the condensers were controlled by a thermostatic bath (TECNAL, Model TE-2000) and the maintenance of the system pressure was made by vacuum pump (TECNAL, Model TE-058).
Qualitative and quantitative analyses
The quantitative and qualitative analyses of the oils were performed by the capillary GC/MS on an Agilent 7890A mass selective (MS) detector system operating at 70 eV using a HP-5MS capillary column (30 m x 0.25 mm x 0.25 mm). Injector and detector temperatures were set at 60 and 325/350 ºC, respectively; the oven temperature was programmed from 60 - 300 ºC in HP-5 column and 60 - 230 ºC.
Compound identification was based on a comparison of the retention indices (determined relatively to the retention times of a series of n-alkanes) and mass spectra with those of authentic samples and/or with literature data (Adams, 2007).
The essential oils and fractions were serially diluted in ethanol (95%) in order to obtain the concentrations of 25.0, 12.5, 6.25, 5.0, 2.5, 1.25 and 0.625 µL/mL. The Mozo strain used as susceptible reference strain was provided by the IPVDF (Instituto de Pesquisas Veterinárias Desidério Finamor). Engorged females R. (B.) microplus were collected from the infested animals, washed with water and dried in paper towel. The average weight of engorging ticks was 0.3 g. These females were incubated at 2728 oC and 7080% relative humidity for two weeks until the egg laying. These eggs provided the larvae used for the larval immersion test - LIT (Ribeiro et al. 2007).
The LIT was conducted by placing approximately 100 embryonated eggs (0.005 g) into pockets (1.0 cm x 1.5 cm) made with TNT fabric. The pockets were incubated at 2728 oC and 7080% relative humidity for 14 days, until the eggs started to hatch. After another 14 days, the pockets containing the larvae ready for testing were immersed for 5 minutes in 10 20 mL of the test solutions. Ethanol (95%) was used as control. After 1 h to allow the solvent to evaporate, the pockets were incubated at 2728 oC and 7080% relative humidity for 48 h and then larvae (alive and dead) were counted to assess the percent mortality. Each treatment contained three replicates.
RESULTS AND DISCUSSION
The citronella and pepper tree essential oils have been widely used as insect repellent. Several studies have been done and the efficacy of the oils against mosquitos and ticks has been demonstrated. Nevertheless, no study has been carried out with the essential oil fractionated by vacuum distillation. In order to prepare the fractions, the essential oils obtained from the aerial parts of C. winterianus and from the fruits and aerial parts of S. molle by steam distillation were submitted to vacuum distillation at constant pressure varying the temperatures. The average yield of each fraction was calculated from the relationship between the fraction volume and essential oils volume. The results are shown in Table 1.
The percentages of each component of the C. winterianus and S. molle essential oils and fractions obtained by vacuum distillation are reported as raw percentages without standardization in the Table 2 and Table 3, respectively. Some compounds not detected in the essential oils and present in the heavy fractions could be formed by thermal degradation or they were detected in these fractions since the main components, more volatiles, were removed in the former fractions.
Laboratory tests were carried out to determine the toxicity of the citronella and pepper tree essential oil fractions on the larvae of the cattle tick R. (B.) microplus by the larval immersion test. In the experiments performed with the citronella essential oil and fractions, the samples presented activity in relation to the control. According to the lethal concentration values (LC) presented in Table 4, the fractions FR1 and FR2 were less active than the total essential oil. Fraction FR3 presented LC values similar to those obtained for the essential oil while fractions FR4 and FR5 proved to be the most active samples. Although the chemical composition of the fractions FR4 and FR5 were different, the activities did not differ significantly.
In the present study, the essential oil of C. winterianus demonstrated activity against the larvae of the cattle tick in concentrations lower than those previously reported (Olivo et al. 2008). The result could be explained by the variability in the components of the essential oil due to environmental factors or by the susceptibility of the strain.
Martins (2006) demonstrated that when tested separately, citronellal and geraniol exhibited higher acaricidal properties compared to citronellol. Nevertheless, in this work, the fractions containing lower amount of citronellal were the most active. The high activity could be enhanced by the presence of higher amount of geraniol. The activity of the fraction FR5 could be attributed to the geraniol and to the presence of sesquiterpenes and diterpenes, which, due to the higher molecular weight, could remain longer in contact with the parasite.
The other essential oil investigated in this work, the pepper tree essential oil, has been previously studied showing antimicrobial and repellent activity (Ferrero et al. 2006, Ferrero et al. 2007; Hayouni et al. 2008, Huerta et al. 2010). In this work, the toxicity of the S. molle essential oil and fractions obtained by the vacuum distillation on larvae of the cattle tick R. (B.) microplus was determined, as shown in Table 5. In relation to fractionation of S. molle essential oil, it could be seen that the less volatile fraction, FR3, presented acaricidal activity, while for the other fractions and the essential oil this activity against the cattle tick was not observed.
Results from this study showed that the less volatile fractions were more active against the tick, which indicated that the contact time of the extract with the larvae was a factor that could influence the toxic effects. It was also observed that the chemical composition of the fractions varied
significantly, which indicated the influence of chemical composition on acaricidal activity. These observations were only possible due to processing by vacuum distillation of essential oils, and thus, obtaining new fraction for the testing of volatile compounds against the cattle ticks.
From the results, it could be concluded that the less volatile essential oil fractions obtained by vacuum distillation were more active against the cattle tick larvae. Especially for the S. molle essential oil, it was observed that a change in the composition caused a significant variation in acaricidal activity. The citronella essential oil fractions presented different behavior against the cattle tick, mainly the fractions FR4 and FR5, when compared with the total essential oil. Results showed that the use of vacuum distillation process could be an alternative in the development of new natural products to be used in the tick control, since the changes in the composition of the essential oils promoted a change in their toxicity against the larvae of R. (B.) microplus.
The authors would like to thank to Programa CYTED, CAPES and CNPq for financial support.
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Received: April 07, 2011;
Revised: October 11, 2011;
Accepted: May 07, 2012.