In vitro efficacy of essential oils with different concentrations of 1,8-cineole against Rhipicephalus (Boophilus) microplus

The aim of this study was to evaluate the acaricidal activity of essential oils from three species of plants with intermediary concentrations of 1,8-cineole against the tick species Rhipicephalus (Boophilus) microplus. For this purpose, five serial concentrations (100.0, 50.0, 25.0, 12.5, 6.2 mg/mL) of essential oils from Mesosphaerum suaveolens (L.) Kuntze, Ocimum gratissimum L. and Alpinia zerumbet (Pers.) B. L. Burtt & R. M. Sm. were used on larval packet and adult immersion tests. The essential oils were analysed by gas chromatography-mass spectrometry (GC/MS) and gas chromatography-flame ionization detection (GC-FID), being detected 35.8, 24.7 and 24.0% of 1.8-cineol in the oils of M. suaveolens, O. gratissimum and A. zerumbet, respectively. The lethal concentration (LC50) of each oil for larvae and engorged females was calculated through Probit analysis. All essential oils showed high efficacy (≥ 95.0%) on engorged females at the 100.0 mg/mL concentration. In regards to larvae, O. gratissimum (LC50 = 11.9 mg/mL) was the most potent, followed by the A. zerumbet (LC50 = 19.7 mg/mL) and the M. suaveolens (LC50 = 51.6 mg/mL) essential oils. These results show that other compounds interfere with 1,8-cineole action.


Introduction
Ticks are parasites with economic importance for bovine and other domestic species in tropical and subtropical countries (MUHAMMAD et al., 2008).Blood spoliation, reduced weight gain, and transmission of Babesia spp.and Anaplasma marginale cause severe economic losses for livestock farmers (GRISI et al., 2014).The control of this parasite often requires the use of intense chemical acaricides that consequently select for resistant populations (RODRÍGUEZ-VIVAS et al., 2006).Acaricide resistance in ticks has become a major problem throughout the world and has been detected in Rhipicephalus (Boophilus) microplus against almost all the registered pesticides indicated for use against this parasite (CASTRO-JANER et al., 2009).Aditionally, the chemical control are costly and contaminate the environment with residues harmful to the hosts and also humans (FREITAS et al., 2005).On the other hand, substances obtained from plants have a low cost, few residual effects and a low incidence of generating resistance (ROSADO-AGUILAR et al., 2010).Essential oils contain a variety of volatile secondary metabolites that are known for their significant role in plant defence mechanisms (NAZZARO et al., 2013).These biodegradable, bioactive oils could be an alternative source for controlling insects, helminths and ticks (BAGAVAN et al., 2009).The terpenes in essential oils are an alternative means to control parasites (ANTHONY et al., 2005).The chemical variability in essential oils and the relationship among the compounds play important roles in the acaricidal activity (SOARES et al., 2016).It is not possible to affirm that the most abundant chemical compounds in an essential oil are responsible for the observed biological effects (GALINDO et al., 2010).Compounds found in small proportions can act as synergists with other minor or major molecules (SOARES et al., 2016).1,8-cineole is an oxygenated monoterpene that acts on cell membranes, mainly through its hydrophobicity (HONÓRIO et al., 2015).This terpene exists, at varied concentrations, in several essential oils with acaricidal activity (PRATES et al., 1998;CHAGAS et al., 2002;SOARES et al., 2016).Studies comparing the essential oils of plants with intermediate concentrations of 1,8-cineole (24 to 35%) are scarce, and understanding of how this compound participates in the activity of natural oils remains obscure.
The aim of the present study was to evaluate the acaricidal activity of essential oils, from aerial parts of two species of the Lamiaceae family (Mesosphaerum suaveolens, Ocimum gratissimum) and one of the Zingiberaceae family (Alpinia zerumbet), with intermediary concentrations of 1,8-cineole against the females and larvae of tick R. (B.) microplus.
The essential oils were extracted at the Unidade de Execução de Pesquisa da Embrapa Meio-Norte, in Parnaíba, Piauí, from the aerial parts of M. suaveolens and from the leaves of O. gratissimum by hydro-distillation using a Clevenger apparatus.Nearly 2 kg of fresh plant was mixed with 3 L of water and boiled for 3 h.A water steam distillation method was used to extract the essential oil from the leaves of A. zerumbet.The essential oils obtained were dried over anhydrous sodium sulphate, filtered, and stored at 4 °C until being tested and analysed at the Embrapa Agroindústria Tropical, in Fortaleza, Ceará, by Gas Chromatography Mass Spectrometry (GC-MS) and Flame Ionization Detector (GC-FID) for determination of their chemical composition (CASTRO et al., 2016).

Essential oil analysis
The GC-MS analyses were carried out on an Agilent 7890B-GC/5977A-MS instrument equipped with a nonpolar VF-5MS fused silica capillary column (30 m × 0.25 mm ID, 0.25 µm film thickness) utilizing a split ratio of 1:30 and helium at 1.5 mL min -1 as the carrier gas.The injector temperature and detector temperature were set at 250 °C.The oven temperature was raised from 70 to 180 °C at 4 °C min -1 and afterwards to 250 °C at 10 °C min -1 .Mass spectra were recorded in a range of mass-to-charge ratio (m/z) between 30 and 450.GC-FID analyses were performed on a Shimadzu GC-2010 Plus chromatograph in the same aforementioned chromatographic conditions except for the carrier gas (hydrogen).The retention indices were determined by the injection of a mixture of C 7 -C 30 homologous n-alkanes (Sigma, St. Louis, MO).The identification of the volatile compounds was achieved through the comparison of the mass spectra recorded with those provided by the spectrometer database (NIST 02-287,324 compounds) along with the retention indices and mass spectra of the literature (ADAMS, 2007;NIST, 2011).The relative content of each constituent was quantified as the normalized peak area detected in the GC-FID chromatogram and expressed as a percentage.

Preparation of the dilutions
For each oil, solutions were prepared at concentrations of 6.2, 12.5, 25.0, 50.0 and 100.0 mg/mL.The negative controls consisted of the solvents used in the essential oil dilutions, which were 50% ethanol and 3% Tween 80.As the positive control, a mixture of 0.18 mg/mL cypermethrin, 0.30 mg/mL chlorpyrifos, and 0.012 mg/mL citronellal (Colosso  , Ouro Fino, São Paulo) was used.This solution was diluted to a 0.125% concentration in ultrapure water.All tests were replicated three times.

Tick preparation
Engorged R. (B.) microplus females were collected from naturally infected cattle, washed with water and then dried with paper towels.A number of the engorged females were kept in an incubator and maintained at 27±1 °C with relative humidity (RH) ≥ 80% until oviposition of the larvae (for the larval packet test) was finished.Acaricide tests had been performed previously that demonstrated the resistance of the tick population to amidinic and pyrethroid compounds.The analyses with ticks were performed at the Unidade de Execução de Pesquisa da Embrapa Meio-Norte, in Parnaíba, Piauí.

Larval packet test
The larval packet test was used following the method of Stone & Haydock (1962) and the Food and Agriculture Organization of the United Nations (FAO, 1971).Approximately one hundred larvae, 14-21 days old, were placed between two filter papers (2×2 cm) impregnated with the appropriate essential oil and concentration, to form a sandwich.Each "sandwich" was introduced to a filter paper envelope and then sealed, identified, and incubated at 27 °C with RH ≥ 80% for 24 h (LEITE, 1988).After this time, larvae, alive and dead, were counted.Ticks showing no movement were considered dead.Each treatment was performed in triplicate.Mortality was calculated from the average of three replicates: % test mortality 100 % control mortality Corrected mortality % 100 % control mortality (1)

Adult immersion test
The adult immersion test of engorged R. (B.) microplus females was conducted according to the method described by Drummond et al. (1973).Engorged females were selected based on their mobility, body integrity, and size (≥ 4.5 mm).The females were then weighed and distributed into ten-specimen groups with similar weights.The weight of engorged females ranged between 170 and 210 mg (BENNETT, 1974).Each group of females was immersed in their appropriate treatment solution for 5 minutes, dried with paper towels, and stored in a chamber at 27 °C and RH ≥ 80% for 18 days.After that period, the egg mass was weighed, transferred to adapted syringes, and incubated for 20 days (27 °C and RH ≥ 80%).The experiment was performed with three replicates for each treatment.
This study was performed with the approval of the Ethics Committee for Animal Experimentation of the Universidade Federal do Maranhão under approval number 23115018061.

Statistical analysis
The lethal concentration of the essential oil for 50% of the population (LC 50 ) of larvae and engorged females was calculated through Probit analysis with GraphPad Prism 7.0 software.An essential oil was considered to be significantly different from another one when the 95% confidence intervals of the calculated LC 50 did not overlap (RODITAKIS et al., 2005).The difference between the concentrations of mortality against larvae and engorged females and the index RO, hatchability and EP were analysed by a paired t-test (p<0.05).

Results and Discussion
The chemical compositions of the tested essential oil samples are presented in Table 1.The chemical composition of the A. zerumbet essential oil was previously determined by Castro et al. (2016).Twenty-three, nineteen and twenty-one components were identified in the essential oils from M. suaveolens, O. gratissimum and A. zerumbet, respectively, representing over 96% of their total compounds.
One of the major constituents in all three species of essential oils was 1,8-cineole.This pure monoterpene caused 100% lethality in R. (B.) microplus larvae (PRATES et al., 1998).On the other hand, 1,8-cineole extracted from Ocimum species and used at a 2% concentration was not an effective acaricidal against R. (B.) microplus (HÜE et al., 2015).This latter result may be due to a low concentration of this compound in the evaluated solution.
1,8-Cineole is present in several essential oils that do and do not have activity against R. (B.) microplus.At a concentration of 10.6% in the Melinis minutiflora essential oil and of 85.8% in Eucalyptus globulus essential oil, the acaricidal action of 1,8-cineole was maximum (100%) on larvae of R. (B.) microplus (PRATES et al., 1998;CHAGAS et al., 2002).A change in the major components of essential oils does not always lead to significate changes in their action on ticks.Minor components of the oil mixture are just as important as major constituents, and their effects must be considered as a reflection of the interaction of all the constituents (AKHTAR et al., 2012).As an example, the essential oil from O. gratissimum contains 24.7% of 1,8-cineole and presents high acaricidal activity on tick females and larvae (Table 2 and 3), however Ocimum canum essential oil, which is 70.2% 1,8-cineole, does not show any action on R. (B.) microplus (HÜE et al., 2015).It is possible that the identified components of essential oils could have antagonistic activity towards each other and consequently reduce the acaricidal action of other major compounds (SOARES et al., 2016).
All three essential oils tested in this study exhibited significant anti-tick activity against R. (B.) microplus.The M. suaveolens essential oil had significantly lower activity (LC 50 = 51.6 mg/mL) on larvae than both the O. gratissimum (LC 50 = 11.9 mg/mL) and the A. zerumbet (LC 50 = 19.7 mg mL -1 ) essential oils.Moreover it was significantly lower (LC 50 = 31.3mg mL -1 ) than the A. zerumbet (LC 50 = 20.7 mg mL -1 ) essential oil on engorged females (Table 4).The presence of a probable antagonistic compound to 1,8-cineole in the M. suaveolens essential oil could justify the reduced larval mortality in relation to the other species.Essential oils has complex mixtures of chemical constituents that may act together synergistically or antagonistically contributing to control of ticks by different action modes (SOARES et al., 2016).The action of the three essential oils was dose-dependent in the two different life stages of the tick.Tables 2 and 3 present the percentages obtained for in vitro action of the three essential oils on the larvae (mortality) and engorged females (egg production index, reduction in oviposition, eggs hatched and efficiency).
In addition to having the lowest LC 50 among the essential oils tested in the present study, the M. suaveolens essential oil, at 100 mg/mL, reduced larvae mortality to 87.7% and had 100% efficacy on engorged females.In Laos, the M. suaveolens essential oil is a strong repellent against the tick Ixodes ricinus.This oil also has 1,8-cineole (16.5-46.6%)and sabinene (15.0-21.2%)as major compounds, which vary in concentration according to the geographic origin of the plants (ASHITANI et al., 2015).Furthermore, the chemical differences in the genotypes of the plant may influence the acaricidal activity against R. (B.) microplus (SOARES et al., 2016).
The O. gratissimum essential oil tested in our study induced high mortality (98.3%) on R. (B.) microplus larvae at a concentration of 5.0% (50 mg/mL) (Table 2).Nevertheless, the essential oil from this plant (undisclosed chemical composition) collected in the Minas Gerais state (Brazil) caused only 74.6% of larvae mortality, even though its concentration was five times higher (25%) (HOCAYEN & PIMENTA, 2013).Ocimum is also known for its large chemotypic variation depending on the geographical origin (HILTUNEN & HOLM, 1999).Environmental factors could support one expressive variation in chemical composition and consequently alter the oil's acaricidal efficacy (HÜE et al., 2015).In regards to engorged females, the O. gratissimum essential oil of this study at 10% (100 mg/mL) achieved 99.9% efficacy.
The proportion of the major compounds of the A. zerumbet essential oil (p-cymene, 1,8-cineole, 4-terpineol) in this study are similar to those reported for this species that greatly inhibited (81.2%) the egg hatch of the parasite Haemonchus contortus (MACEDO et al., 2013).The larvicidal effect of essential oils from A. zerumbet and O. gratissimum (100 mg/mL) was similar to that induced by chemical acaricide (0.18 mg/mL cypermethrin, 0.30 mg/mL chlorpyrifos, and 0.012 mg/mL citronellal).
In engorged females, the three essential oils, at 100 mg/mL, greatly reduced oviposition (ranging from 95.5 to 100.0%) and hatchability (0.0 a 0.8%), exhibiting an average efficiency that ranged from 99.9 to 100%, but there was no significant difference among them at this concentration (p > 0.05).This action on engorged females was similar to that induced by the positive control (Table 3).
The susceptibility of adult Rhipicephalus spp. to essential oils generally appears to be substantially lower than that of the larvae (ELLSE & WALL, 2014).As an example, in this study, the essential oils from A. zerumbet and O. gratissimum obtained an
efficacy value of 99.9 to 100.0% against engorged females when used at a concentration of 100 mg/mL, while similar values of larvae mortality (98.3 to 100%) were observed at concentrations 50% lower (50 mg/mL).However, opposite results were obtained when the M. suaveolens essential oil was evaluated, which had a higher activity against the engorged females (100%) than the R. (B.) microplus larvae (87.7%) at 100 mg/mL.This variation in activity is probably related to each oil's unique mode of action against the specific life stage of the tick (SOARES et al., 2016).
In this study, the first point of contact of the essential oils is the cuticle of engorged females and larvae (DRUMMOND et al., 1973;LEITE, 1988).When crossing the integument of the tick, the substances can reach the haemolymph and act on the internal organs (REMEDIO et al., 2015).Certain substances may affect the salivary glands, consequently impairing the process of concentrating ingested blood and thus hindering the effective absorption of nutrients by the ticks (REMEDIO et al., 2016).There is an important correlation between the tick's digestive and reproductive system and, thus, digestive problems can damage their reproductive capacity (REMEDIO et al., 2016).The cattle tick has a high biotic potential (OLIVER, 1989), therefore products and substances that act on their reproductive system are of great interest in the control of these populations (OLIVEIRA et al., 2016).The ovary of R. (B.) microplus consists of an epithelial cell wall and oocytes (germ cells) that are at different developmental stages (OLIVEIRA et al., 2005).An injury to the oocytes decreases the female's capacity to produce viable eggs and descendents.A high reduction in oviposition and hatchability resulted in a high efficiency of the essential oils from A. zerumbet, O. gratissimum and M. suaveolens, at 100 mg/mL, against engorged females of R. (B.) microplus (Table 3).
Functional impairment of the reproductive system of ticks is usually a consequence of significant morphophysiological alterations that have been detected in females exposed to andiroba oil (Carapa guianensis Aubl.) (VENDRAMINI et al., 2012) and to neem oil (Azadirachta indica A. Juss.) (REMEDIO et al., 2015).Some extracts of plants, when used to treat females in the beginning or in the middle of the engorgement process, are more efficient in the control of this parasite.Thus, the substances can penetrate the cells more easily and act in their interior (OLIVEIRA et al., 2016).It is reasonable to assume that the efficacy of the three essential oils in this study would be higher in early stages of engorged females even at lower concentrations.
The tick has a central nervous system that is known as synganglion (ROMA et al., 2012).Neural morphological damage resulting from a degenerative process would be capable of affecting nerve impulse transmission in ticks, impairing locomotion and feeding mechanisms.Some oils that originate from plants promote such morphological changes (ROMA et al., 2013).Neurotoxic action can be caused also by oils and compounds that inhibit acetylcholinesterase (AChE), an enzyme essential for the transmission of action potentials (MILLS et al., 2004;LÓPEZ & PASCUAL-VILLALOBOS, 2010).The monoterpenoid 1,8-cineole inhibit AChE activity from insects (ABDELGALEIL et al., 2009) and is one of the main compounds of the three essential oils evaluated in this study whose efficacy on larvae and females was demonstrated.This result indicates the possible action of these three essential oils on the nervous system of the tick.
The possibility of reduction in risks of contamination of food and the environment by alternative products is a worldwide desire (ADENUBI et al., 2016).However, more studies should be performed to develop formulations that protect this oils and your active compounds from environmental degradation.These results, demonstrated that the essential oils evaluated may be a viable alternatives to synthetic acaricides.

Conclusion
All three essential oils evaluated show a high efficacy against engorged females at their highest concentration and produced similar results to those induced by chemical acaricide.In regards to larvae, O. gratissimum was the most efficient, followed by the A. zerumbet and the M. suaveolens essential oils.Our results indicated that minor compounds interfere on the action of 1,8-cineole present in essential oils.

Table 1 .
Comparative chemical composition of essential oils from Mesosphaerum suaveolens, Ocimum gratissimum and Alpinia zerumbet.Kovats index displayed by compounds in RTX column; b KI lit : Literature data (ADAMS, 2007; NIST, 2011); c Peak relative areas determined in the GC-FID chromatogram; *Data reported by Castro et al. (2016).
a KI: