Acaricidal activity of essential oils of Cinnamomum zeylanicum and Eremanthus erythropappus, major compounds and cinnamyl acetate in Rhipicephalus microplus

Braz J Vet Parasitol 2021; 30(3): e009221 | https://doi.org/10.1590/S1984-29612021070 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Acaricidal activity of essential oils of Cinnamomum zeylanicum and Eremanthus erythropappus, major compounds and cinnamyl acetate in Rhipicephalus microplus


Introduction
Rhipicephalus microplus (Canestrini, 1888), also known as the cattle tick, can cause several types of harm to livestock. In addition, this species is a limiting factor for the productive success of cattle raising (Labruna & Machado, 2006;Furlong et al., 2007). The annual economic losses caused by this ectoparasite in Brazil can reach US$ 3.24 billion (Grisi et al., 2014), while the annual losses estimated worldwide range from US$ 22 to 30 billion (Lew-Tabor & Rodriguez Valle, 2016).
Currently, the main tick control method consists of use of synthetic acaricides. These provide relatively fast and cost-effective suppression of populations and are easily available on the market (Abbas et al., 2014). However, continuous use of these products can lead to issues such as environmental contamination, residues in milk and meat, and development of resistant tick populations (Graf et al., 2004). These issues have motivated the development of new tick control technologies (Zaman et al., 2012).
Studies have shown that essential oils (EOs) extracted from aromatic plants are an ecologically sustainable (eco-friendly) alternative with potential for use in tick control. Among the characteristics of EOs that favor their use, it is worth emphasizing the fact that they derive from renewable resources, delay the selection of resistant populations because they present a complex mixture of compounds, and present lower risk of environmental and animal harm because of their high biodegradability rates and low toxicity in mammals (Borges et al., 2011;Madzimure et al., 2011;Liu et al., 2017).
Species of the Asteraceae and Lauraceae families are among the plants from which EOs with potential for tick control can be extracted. Cinnamomum zeylanicum Blume (cinnamon) (synonym -Cinnamomum verum) is a plant of the Lauraceae family from which the essential oil has been successfully tested for biological activity against bacteria (Mishra et al., 2008), fungi (Carmo et al., 2008), insects (Yang et al., 2005) and ticks (Monteiro et al., 2017;Jyoti et al., 2019;Nwanade et al., 2021). (E)-cinnamaldehyde is the main constituent in the essential oil extracted from C. zeylanicum bark. This phenylpropanoid presents activity against bacteria and arthropods (Shen et al., 2012;Senra et al., 2013;Novato et al., 2015).
Studies focused on investigating structural modifications in substances extracted from EOs, based on changes in functional groups, have been conducted in order to enhance the biological activity or increase the biosafety of these compounds (Kim et al., 2015). These changes include the acetylation process based on introduction of an acetate group. This process, which replaces the hydroxyl group (-OH) with the acetate group, provides molecules with greater cuticular permeability, thus facilitating absorption into the arthropod (Lanusse & Prichard, 1993). In addition, it is less toxic to mammals (André et al., 2016(André et al., , 2017, which makes its acaricidal use safer both for animals and for the environment. According to some studies, the acetylation process can increase the activity of these compounds on bacteria, insects and ticks (Mathela et al., 2010;Scotti et al., 2014, Ramírez et al., 2016. The aims of the present study were to chemically characterize the EOs extracted from E. erythropappus stems and C. zeylanicum bark; and to evaluate the acaricidal activity of these oils, their major compounds and the (E)cinnamaldehyde acetylated derivative (= cinnamyl acetate) against unfed larvae and engorged females of R. microplus.

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Botanical compounds against ticks Chemical analysis of essential oils The EOs were subjected to qualitative analysis of their chemical composition through gas chromatography coupled with mass spectrometry (GC/MS) using the Shimadzu GC-2010 device (quadrupole) with the following characteristics: electron impact at 70 eV; RTX-5MS methylpolysiloxane column (30 m x 0.25 mm x 0.25 μm; Restek®); 1:100 split injection mode; helium carrier gas flow rate of 1.00 mL/min; and the following temperatures: injector 250 °C, transfer line 250 °C and ion source 230 °C. The gas chromatography oven was programmed as follows: initial temperature of 70 °C; heating ramp of 4 °C/min up to 180 °C; and temperature increase rate of 10 °C/min up to 250 °C at the end of the run (34.5 min).
The quantitative analysis applied to the chemical composition of the EOs was performed through gas chromatography coupled to flame ionization detection (CG/FID) using a Shimadzu GC-2010 Plus spectrometer set up as follows: RTX-5 methylpolysiloxane column (30 m × 0.25 mm × 0.25 μm); split injection mode (1:30); nitrogen carrier gas flow rate of 1.00 mL/min; injector temperature 250 °C; and detector temperature 280 °C. The gas chromatography oven was programed as in the GC/MS analysis.
Percentages of the chemical constituents of the EOs were calculated based on the integral area of their respective peaks, in comparison with the total area of all sample constituents. Several essential oil constituents were identified by visually comparing their mass spectra with those reported in the literature (Bohlmann et al., 1998;Adams, 2009) and with the spectra provided by the equipment database (NIST08), as well as by making comparisons with the retention indices available in the literature (Adams, 2009;El-Sayed, 2018). An n-alkane standard solution (C7-C30) was injected under the same chromatographic conditions as that of the sample, in order to find retention rates, as described by van Den Dool & Kratz (1963).

Essential oils and compounds
Eremanthus erythropappus essential oil, obtained by means of steam distillation from the stem of this plant species cultivated in the state of Minas Gerais (MG), was kindly supplied by Atina -Ativos Natural -Ltda (Pouso Alegre, MG, Brazil). C. zeylanicum essential oil was acquired commercially from Laszlo® (Belo Horizonte, MG, Brazil); the oil of this plant, which originates from Sri Lanka, was obtained by steam distillation of the plant bark. The α-bisabolol (95% purity) was kindly provided by Citróleo Indústria e Comercio Ltda (Torrinha, SP, Brazil), while (E)-cinnamaldehyde and cinnamyl alcohol (98% purity) (used for production of cinnamyl acetate) ( Figure 1) were purchased from Sigma-Aldrich (St Louis, MO, USA).

Acetylated derivative preparation
The (E)-cinnamaldehyde acetylation process was performed at the Natural Products Laboratory of the Federal Rural University of Rio de Janeiro (UFRRJ), based on the descriptions of Oliveira et al. (1999). To this end, cinnamyl alcohol (4.44 g), acetic anhydride (8.88 mL) and pyridine (6.66 mL) were added to a 25 mL flask. Reagents were placed under stirring with the aid of a magnetic stirrer equipped with a stirring plate, for 24 h. The product from the reaction was added with chloroform. The residual solution was washed with hydrochloric acid (10%) until all pyridine had been removed from the system through soluble salt formation.

Acetylation product confirmation
Nuclear magnetic resonance (NMR) (1D) spectrum analyses were performed using a Bruker AVANCE II 11.5 T spectrometer (500 MHz for 1 H and 125 MHz for 13 C) at the analytical center of the Chemistry Institute, Federal Rural University of Rio de Janeiro for product confirmation purposes. Tetramethylsilane (TMS) was used as the internal standard for chemical shift reference. Chemical shifts (δ) were expressed in parts per million (ppm), while coupling constants (J) were expressed in Hertz (Hz). Deuterated chloroform (CDCl 3 ) was used as the solvent in the NMR analysis.

Ticks
Ticks were collected from naturally infested cattle on farms in Minas Gerais, Brazil, and were provided by the Laboratório de Parasitologia da Empresa Brasileira de Pesquisa Agropecuária (Embrapa Gado de Leite), located in the municipality of Juiz de Fora, state of Minas Gerais, Brazil.
Half of the R. microplus engorged females were used to perform the adult immersion test (AIT), while the other fraction was stored in a climatized chamber with controlled temperature and relative humidity (27 ± 1 °C and RH of 80 ± 10%) for 15 days until oviposition. After this stage, the egg mass was weighed into 200 mg aliquots and packed into syringes with a closed distal end, which were sealed using cotton wool. These eggs were then kept under the same aforementioned temperature and humidity conditions. Between 15 and 21 days after hatching, the larvae were used to perform LPT. The same tick population was used for the AIT and LPT tests on the same oil or compound.
The LPT proposed by Stone & Haydock (1962) and adapted by Monteiro et al. (2012) was used to assess the activity of the EOs and their major compounds. Approximately 100 unfed larvae were placed in the center of a filter paper (6 cm x 6 cm; Whatman no. 1), which was then folded in half and closed at the sides using binder clips. Next, each side of the filter paper was moistened with 90 µL of test solutions (180 µL in total). A control group was also formed (70% or 50% ethanol), and 10 replications were performed for each group.
The packets were then stored in a B.O.D. incubator at 27 ± 1 °C and RH of 80 ± 10% for 24 h. After this period, the living and dead larvae were counted.

Adult Immersion Test (AIT)
For the AIT, groups of 10 engorged females (each female = 1 experimental unit) presenting homogeneous weights (p > 0.05) were immersed at concentrations of 2.5, 5.0, 10.0, 20.0, 40.0 and 60.0 mg/mL (10 per concentration) for 5 min. Concentrations were selected through preliminary tests and dimethyl sulfoxide (DMSO) at 3% (water + DMSO, v/v) was used as the solvent. After immersion, each female was weighed individually and placed on a Petri dish (6 x 6 cm) for oviposition. The groups were kept in a climate-controlled chamber under the aforementioned conditions for reproductive biology assessment. The following biological parameters were evaluated: female weight before oviposition (soon after treatment), egg mass weight (15 days after treatment) and larval hatching (21 days after egg mass weighing). From these values, the percentage control was calculated as described by Drummond et al. (1973).

Statistical analysis
The statistical analyses conducted on both tests were carried out using the Biostat 5.3 software (Ayres et al., 2007). Treatments were compared by means of analysis of variance (ANOVA) followed by Tukey's test. Non-normally distributed data were subjected to the Kruskal-Wallis test followed by the Student-Newman-Keuls test. Probit analysis was performed using the R software (version 3.5.3, 2019) to enable calculation of the lethal concentration 50 (LC 50 ).

Components
Cinnamomum

Larval packet test
The essential oil extracted from C. zeylanicum bark at the concentration of 1.25 mg/mL resulted in a 64.0% tick mortality rate, with a difference (p < 0.05) in comparison with control groups (0.0%). The mortality rate was > 95% from the concentration of 2.0 mg/mL and reached 100.0% at the concentration of 2.5 mg/mL (Table 2).
(E)-cinnamaldehyde resulted in mortality of larvae with differences (p < 0.05) in relation to the control groups, starting from the concentration of 1.25 mg/mL (29.2%). Larval mortality > 95% was observed from the concentration of 2.5 mg/mL. However, only the highest concentration (5.0 mg/mL) resulted in a 100.0% larval mortality rate. Cinnamyl acetate at the concentration of 2.0 mg/mL led to 44.0% larval mortality, which was different from the mortality rate observed for the control groups (p < 0.05). At the highest concentrations (5.0 and 10.0 mg/mL), mortality was 99.0 and 100.0%, respectively (Table 2).
Eremanthus erythropappus essential oil resulted in mortality > 90% from the concentration of 2.0 mg/mL and reached 100.0% larval mortality at the concentration of 2.5 mg/mL, with differences (p < 0.05) in comparison with the control groups (0.0%). The α-bisabolol at the concentration of 2.0 mg/mL led to a larval mortality rate of 32.1%, which was different (p < 0.05) from that observed in the control groups. Mortality rates > 95% were observed starting from the concentration of 5.0 mg/mL (96.7% mortality), and reached 100.0% mortality at the concentration of 10.0 mg/mL ( Table 2).

Acetylation confirmation
The hydrogen ( 1 H NMR) and carbon-13 ( 13 C NMR) nuclear magnetic resonance (NMR) spectra that were used to confirm the acetylated product are shown in Figure 2. The final product was weighed and presented a mass of 5.25 g, with 90% yield.

Adult immersion test
For C. zeylanicum EO, female weight before oviposition did not differ between treatments (p > 0.05). This EO did not result in differences (p > 0.05) in egg mass weight between treatments and the control group. The larval hatching rate for the treatment with the lowest concentration (2.5 mg/mL) was 16.6 ± 10.1% (p < 0.05), whereas in the control group, larval hatching was 85.4 ± 7.5%. At the highest concentrations (40.0 and 60.0 mg/mL), no larvae hatched. The percentage control was > 90% starting from the 5.0 mg/mL concentration and reached 99 and 100% in the treatments with 10.0 and 40.0 mg/mL, respectively (Table 4).     The egg mass weight of the groups treated with (E)-cinnamaldehyde ranged from 101.1 ± 7.5 to 0.0 mg, which was different (p < 0.05) from the value recorded for the control group (145.0 ± 13.7 mg). Similarly, larval hatching ranged from 28.5 ± 24.7 to 9.4 ± 1.8% in the treated groups (p < 0.05) and was 96.3 ± 2.4% in the control group. The percentage control was 73.4% at the lowest concentration (2.5 mg/mL) and reached 100.0% at the concentration of 20.0 mg/mL (Table 4).
No difference in mean egg mass weight was observed between engorged females in the control and groups treated with cinnamyl acetate (p > 0.05). With regard to larval hatching, only the three highest concentrations (20.0, 40.0 and 60.0 mg/mL) resulted in values lower (58.1 ± 28.6, 58.0 ± 25.1 and 56.6 ± 8.2%; p < 0.05) than that observed in the control group (80.1 ± 9.4%). None of the treatments resulted in percentage control > 50%, such that this reached a maximum of 42.6% at the concentration of 20.0 mg/mL (Table 4).
For E. erythropappus EO, starting from the concentration of 10.0 mg/mL, a reduction (p < 0.05) in egg mass weight (21.4 ± 26.2 mg) was observed, in comparison with the control group (67.7 ± 22.2 mg). Regarding larval hatching, there was a difference (p < 0.05) between the control (84.2 ± 12.3%) and treated groups, starting from the concentration of 5.0 mg/mL, with values ranging from 39.7 ± 32.0 to 0.0%. The percentage control at the 2.5 mg/mL concentration was 52.4%, and reached 100.0% at the highest concentration (60.0 mg/mL) (Table 4).

Discussion
The essential oil extracted from cinnamon bark that was investigated in the present study resulted in LC 50 of 0.94 mg/mL against larvae. A similar result was observed by Jyoti et al. (2019), who evaluated the activity of the essential oil of C. zeylanicum bark (with 64.4% (E)-cinnamaldehyde) on R. microplus larvae, and observed LC 50 of 0.86 mg/mL. The small differences found may be related to the chemotype of the plant, unequal percentages and chemical constituents, and to different strains of R. microplus.
For engorged females, the present study found a percentage control of 99.8% at the concentration of 20.0 mg/mL. No reports on the activity of cinnamon bark essential oil against R. microplus engorged females were found in the literature. However, Monteiro et al. (2017) reported a percentage control of 27.3% against R. microplus engorged females that were treated with essential oil (with 4.0% (E)-cinnamaldehyde) extracted from C. zeylanicum leaves at the concentration of 25.0 mg/mL. Santos et al. (2017) reported 100.0% effectiveness among R. microplus engorged females exposed to commercial cinnamon essential oil (with 41.27% (E)-cinnamaldehyde) at the concentration of 100.0 mg/mL. Thus, both the data from the present study and the data in the literature indicated that the percentage of this phenylpropanoid in the chemical composition of cinnamon EO had significant influence on its acaricidal activity. This is emphasized by the results regarding activity in the tests with (E)-cinnamaldehyde alone, which showed LC 50 of 1.38 and 1.50 mg/mL for larvae and engorged females, respectively. In the present study, we observed a mortality of 95.3% of the larvae treated at a concentration of 2.5 mg/mL of (E)-cinnamaldehyde, corroborating the results found by Senra et al. (2013), which in the same concentration observed a mortality of 99.2%. In future studies on cinnamon oil for tick control, samples extracted from bark, which is rich in this phenylpropanoid, should be prioritized.
R. microplus larvae treated with E. erythropappus EO (with 78.41% α-bisabolol) at the concentration of 2.0 mg/mL presented a mortality rate > 90.0% in the present study. Diniz (2014) evaluated the activity of four Siparuna guianensis EOs and observed that two samples containing the sesquiterpene α-bisabolol (> 62.6%) presented higher activity against R. microplus unfed larvae and engorged females than two samples that did not have this compound in their chemical composition. El-Moneim et al. (2012) evaluated the action of Chamomilla recutita (L.) Rauscher essential oil, which is rich in α-bisabolol (35.2%), on Tetranychus urticae mites and observed a 100.0% mortality rate at 4.0% concentration. These results indicate that the presence of the sesquiterpene α-bisabolol is a major factor for the high acaricidal activity observed in these EOs. According to Paluch et al. (2009), sesquiterpenes produced by different plant families can be used to affect the behavior of some arthropods and even kill them. The action of sesquiterpene-rich EOs against ticks has previously been recorded in relation to plants belonging to the families Araucariaceae, Cupressaceae (Lebouvier et al., 2013), Asteraceae (Ribeiro et al., 2011;Lage et al., 2014), Lamiaceae (Facey et al., 2005) and Winteraceae (Ribeiro et al., 2008).
The activity of pure α-bisabolol against R. microplus unfed larvae and engorged females was first demonstrated in a study conducted by Diniz (2014), whose results showed a larval mortality rate of 98.9% at the concentration of 5.0 mg/mL and a percentage control of 98.1% among engorged females treated at the concentration of 40.0 mg/mL (Diniz, 2014). The present study recorded larval mortality of 96.7% for the pure α-bisabolol concentration of 5.0 mg/mL, and this sesquiterpene resulted in percentage control of 100.0% at the concentration of 20.0 mg/mL against engorged females. The different results observed in relation to engorged females may be associated with the solvent used. Diniz (2014) used Tween80 as the solvent, whereas DMSO was used in the present study. Studies have shown that certain types of solvent can enhance the effect of EOs , and there are data in the literature demonstrating that certain surfactants can reduce the activity of essential oils (Li et al., 2017). In that regard, some studies have shown that DMSO, being hydrophilic, is capable of penetrating cell membranes and carrying the toxic agent with it (Gorman & Dordick, 1992;Du et al., 2004).
A comparison between the LC 50 values of the EOs and their major compounds (cinnamon essential oil vs. (E)-cinnamaldehyde and candeia essential oil vs. α-bisabolol) demonstrated that these EOs showed higher activity against R. microplus unfed larvae than their major compounds alone. The candeia essential oil also showed higher activity against engorged females, in comparison with α-bisabolol. The higher activity of EOs may be due to synergistic or additive effects resulting from the association between major compounds and other molecules found in the cinnamon and candeia EOs, which can enhance their acaricidal activity. Monteiro et al. (2017) evaluated a chemotype of the EO of C. verum and its major component, benzyl benzoate, against R. microplus larvae and also observed that the EO showed better activity. Costa-Júnior et al. (2016) observed the activity of the EO of Lippia gracilis Schauer and the monoterpene thymol against two strains of R. microplus (unfed larvae) and observed that, in both strains, the EO resulted in greater activity than the monoterpene thymol.
The (E)-cinnamaldehyde used in the present study showed higher activity than α-bisabolol; therefore, only this phenylpropanoid was used to produce the acetylated derivative (cinnamyl acetate). Cinnamyl acetate presented lower acaricidal activity than (E)-cinnamaldehyde in tests conducted using unfed larvae, with LC 50 of 2.31 and 1.38 mg/mL for the larvae. The difference in acaricidal activity was higher in tests conducted using engorged females, since the highest cinnamyl acetate concentration (60.0 mg/mL) resulted in percentage control of only 36%, while (E)-cinnamaldehyde at a concentration of 20.0 mg/ml resulted in percentage control of 100.0%. Lee et al. (2019) compared the acaricidal activity of cinnamon bark essential oil with that of (E)-cinnamaldehyde and cinnamyl acetate against Dermanyssus gallinae (De Geer) and found that cinnamyl acetate was the only substance that did not have repellent and acaricidal effects on these mites. Novato et al. (2018) observed that carvacrol, thymol and eugenol presented higher activity against R. microplus unfed larvae than their acetylated derivatives. However, there is controversy about the enhancement of acetylated compounds for application to ticks. According to Ramírez et al. (2016), carvacrol showed lower activity against R. microplus unfed larvae and engorged females than its acetylated derivative (carvacrol acetate).
All the compounds analyzed in this study significantly reduced the viability of eggs of R. microplus engorged females. Other studies evaluated the activity of different phytochemicals on engorged females and also reported significant reductions of egg viability. Marchesini et al. (2020) observed that Jambu extract and spilanthol reduced the egg viability of R. microplus, and the same was observed for Baccharis dracunculifolia EO and nerolidol (major compound) (Lage et al., 2014) and Lippia triplinervis EO rich in thymol and carvacrol (Lage et al., 2013), against cattle tick. This reduction in egg viability may be related to action by this EO and its compounds alone, in organs related to the reproductive biology of engorged females, such as the ovaries and Gené's organ. Studies have shown that use of pure constituents of EOs such as thymol and semi-synthetic compound acetylcarvacrol led to a number of morphophysiological changes in engorged females' oocytes, with damage to the nucleus and germinal vesicle, cytoplasm vacuolation, membrane rupture and deformation, and a reduced number of yolk granules (Matos et al., 2014;Konig et al., 2019). In addition, a recent study found that the monoterpene thymol had deleterious action on Gené's organ (Matos et al., 2020). Future studies focused on investigating the action of cinnamon and candeia EOs, as well as the action of (E)-cinnamaldehyde and α-bisabolol on these organs, based on use of morphological analysis tools, could assist in achieving better understanding of the mechanisms capable of reducing the viability of the eggs of engorged females.
We conclude that C. zeylanicum and E. erythropappus EOs, as well as their major compounds, showed acaricidal activity on R. microplus unfed larvae and engorged females, while cinnamyl acetate showed low acaricidal activity. In addition, the EOs showed greater activity than their major compounds. The results suggest that some plant compounds are candidates for use as pesticides against green ticks. Further studies to assist in better understanding the possible modes of action of these EOs and their major compounds against R. microplus, as well as tests to assess efficacy against ticks and clinical safety for cattle, are necessary.