Parasitol Res

DOI 10.1007/s00436-017-5604-x

ORIGINAL PAPER

Evaluation of the acaricidal activity of thymol incorporated

in two formulations for topical use against immature stages
of Rhipicephalus sanguineus sensu lato (Latreille, 1806)
(Acari: Ixodidae)
Camila Delmonte 1 & Paula Barroso Cruz 2 & Viviane Zeringóta 2 & Valéria de Mello 3 &
Felipe Ferreira 3 & Maria da Penha Henriques Amaral 3 & Erik Daemon 1

Received: 10 May 2017 / Accepted: 27 August 2017

# Springer-Verlag GmbH Germany 2017

Abstract The objective of this study was to assess, for the
first time, the in vitro acaricidal activity of two topical formu-
lations containing thymol, on immature stages of
Rhipicephalus sanguineus sensu lato. For this purpose, two
base formulations were prepared: an oil-in-water (O/W) emul-
sion and a hydroalcoholic solution, containing different thy-
mol concentrations (0.5 to 20 mg/mL). We used the larval
packet test for non-engorged larvae and nymphs, and the im-
mersion test for engorged larvae and nymphs. For emulsion, a
mortality rate of 94.2% was achieved at 0.75 mg/mL in non-
engorged larvae. For engorged larvae, there was 95.0% mor-
tality at 5.0 mg/mL. Non-engorged nymphs showed 83.3%
mortality at 2.5 mg/mL, and for engorged nymphs, 86.0%
mortality was verified at 5.0 mg/mL. For the hydroalcoholic
solution, the mortality found for non-engorged larvae was
88.1% at 2.5 mg/mL. For engorged larvae, the highest mor-
tality was 25.0% at 20 mg/mL; non-engorged nymphs had
91.0% mortality at 1.0 mg/mL and for engorged nymphs;
the maximum value verified was 18.3% mortality at 20 mg/
mL. Preliminary stability tests were carried, and the
hydroalcoholic solution remained stable under all the
* Camila Delmonte
camila_delmonte@hotmail.com
1
Programa de Pós Graduação em Ciências Biológicas -
Comportamento e Biologia Animal, Universidade Federal de Juiz de
Fora UFJF, Juiz de Fora, Minas Gerais, Brazil
2
Programa de Pós Graduação em Ciências Veterinárias, Departamento
de Parasitologia Animal, Universidade Federal Rural do Rio de
Janeiro, UFRRJ, Seropédica,, Rio de Janeiro, Brazil
3
Programa de Pós Graduação em Ciências Farmacêuticas,
Universidade Federal de Juiz de Fora, UFJF, Juiz de Fora, Minas
Gerais, Brazil
conditions analyzed. The O/W emulsion showed signs of early
instability at the concentration of 5.0 mg/mL. The results ob-
tained indicate that the acaricidal activity of thymol, when
included in the proposed formulations, was enhanced against
non-engorged larvae with topical treatment in comparison
with data in the literature. Although there were variations in
toxicity between the different stages, these formulations are
promising for future therapeutic use.
Keywords Brown dog tick . Monoterpene . Larvae .
Nymphs . Emulsion . Hydroalcoholic solution
Introduction
Rhipicephalus sanguineus sensu lato (Latreille, 1806), the
brown dog tick, is widely distributed in the world (Labruna
2004; Guglielmone et al. 2006). The control of this ectopara-
site should take into account that it is a three-host tick, which
means that each stage of its life cycle must feed on a host, and
that oviposition and molts are made in the environment.
Besides the direct damage caused, such as blood spoliation,
discomfort, and allergic reactions, it can also act as a vector for
dissemination of bacteria and protozoa to dogs and humans
(Dantas-Torres 2008). In Brazil, it is considered a potential
vector of human infection by the bacterium Rickettsia
rickettsii (Serra-Freire et al. 2011).
The taxonomic status of this species still lacks consensus.
In recent years, evidence of genetic, morphological, and epi-
demiological differences of individuals from different regions
of the world has been reported, and based on the difficulty of
determining the neotype R. sanguineus sensu stricto, it has
been established that the most suitable denomination for

individuals of this complex is Rhipicephalus sanguineus
sensu lato (s.l.) (Nava et al. 2015).
Control of these parasites is usually by applying synthetic
chemical substances. However, the indiscriminate use of these
substances causes selection of resistant tick populations, in-
toxication of animals and their handlers, and possible contam-
ination of the environment with chemical wastes (Chagas
2004; Coles and Dryden 2014). Since the relationship be-
tween humans and domestic animals has been getting increas-
ingly close, with dogs often considered as members of the
family, the importance of parasite control and the use of safe
chemicals is emphasized, in order not to compromise the
health of pets and their guardians. These drawbacks have re-
sulted in growing demand for safer acaricides, in turn,
prompting research into substances derived from plants
(Chagas, 2004; Ellse and Wall 2014; Regnault-Roger and
Philogène, 2008; Borges et al. 2011).
Among the substances of plant origin already tested for this
purpose is thymol, an aromatic monoterpene initially isolated
from plants of the family Lamiaceae. This molecule has
shown promising results regarding its potential against vari-
ous species of ticks (Monteiro et al. 2010; Mendes et al. 2011;
Daemon et al. 2012; Matos et al. 2014; Araújo et al. 2015;
Novato et al. 2015).
For a chemical substance, whether natural or artificial, to be
used for therapeutic purposes, it is often necessary to include it
in a formulation that makes its application feasible and does
not impair its efficacy (York 2005). Therefore, the aim of this
study was to assess the in vitro acaricidal activity of thymol,
incorporated in two formulations for topical use, against im-
mature stages of R. sanguineus s.l., along with the formula-
tions’ short-term stability.
Material and methods
Study location
The formulations were prepared, and their stability was ana-
lyzed in the Laboratório de Farmacotécnica da Universidade
Federal de Juiz de Fora, Minas Gerais, Brazil. The in vitro
tests on ticks were performed in the Laboratório de
Artrópodes Parasitos of the same University.
Origin of the ticks
The R. sanguineus s.l. larvae used came from a colony main-
tained by an artificial infestation of rabbits (Oryctolagus
cuniculus Linnaeus, 1758—New Zealand x California cross),
according to the method proposed by Neitz et al. (1971). The
engorged female ticks were kept in a climate-controlled cham-
ber (27 ± 1 °C and RH 80 ± 10%) for oviposition. After
15 days, the eggs were weighed into aliquots of 200 mg,
Parasitol Res
placed in plastic syringes with the distal end cutoff, sealed
with hydrophilic cotton, and labeled for identification. The
syringes were kept under the same temperature and humidity
conditions, and the larvae were used in the tests 15–25 days
after hatching. The unengorged nymphs, derived from
engorged larvae, were tested 15 days after ecdysis and the
engorged larvae and nymphs, obtained by artificial infestation
on rabbits, were tested on the day they dropped off the host.
Formulation development
Two base formulations were prepared, one an oil-in-water
(O/W) emulsion and the other a hydroalcoholic solution.
The thymol utilized in the formulations was obtained from
Sigma-Aldrich, with purity ≥ 99%. The O/W emulsion was
formulated with the following raw materials, identified ac-
cording to the International Nomenclature of Cosmetic
Ingredients (INCI): cetearyl alcohol, sodium laureth sulfate,
metylparaben, propylparaben, glycerin, grape seed oil, and
water. For preparation, each constituent was separated accord-
ing to its solubility in water or oil. The aqueous phase was
heated to 80 °C and the oil phase to 75 °C, and then the
aqueous phase was added in the oil phase and emulsified with
a mortar and pestle until completely homogenized. The
hydroalcoholic solution contained glycerin, methylparaben,
and ethanol and was prepared by mixing the components at
room temperature in a graduated glass beaker. Twenty-four
hours after preparation, the samples were evaluated regarding
homogeneity and organoleptic characteristics, to identify any
instability. In both formulations, the thymol, dissolved in ab-
solute ethanol q.s. (sufficient quantity), was added after prep-
aration of the base formula.
Bioassays
For the in vitro tests, 18 groups were formed, nine for the emul-
sion and nine for the hydroalcoholic solution: emulsion (or solu-
tion) control, and emulsion (or solution) with thymol at 0.5, 0.75,
1.0, 1.25, 2.5, 5.0, 10.0, and 20.0 mg/mL. These concentrations
were chosen based on tests previously carried out with thymol on
R. sanguineus s.l. at concentrations ranging from 2.5 to 20 mg/
mL (Daemon et al. 2009; Monteiro et al. 2009; Daemon et al.
2012; Senra et al. 2013), and lower thymol concentrations were
also tested to assess possible enhancement of the acaricidal effect
caused by the interaction of thymol with the adjuvants in the
formulations. The larval packet test was used for the unengorged
phases, as proposed by Stone and Haydock (1962) and adapted
by Monteiro et al. (2012). Approximately 100 larvae were placed
on sheets of filter paper (6 × 6 cm), which were then folded and
sealed with three binder clips. Then each side of the filter paper
envelope was moistened with 90 μL of the formulation to be
tested, and the envelopes containing the ticks were placed in a
climate-controlled chamber (27 ± 1 °C and RH 80 ± 10%). Each
Parasitol Res
envelope corresponded to a sample unit, and each treatment was
repeated ten times. The envelopes were opened after 24 h to count
the number of live and dead larvae, using a vacuum pump at-
tached to a hose to collect the live ones. The average mortality
was expressed as a percentage, according to the following for-
mula: mortality (%) = (total dead larvae/total of larvae) × 100.
The same method was used for the unengorged nymphs,
except only five nymphs were placed in each packet.
The engorged larvae were submitted to the immersion test,
as proposed by Drummond et al. (1973). Larvae recovered
from the artificial infestation of rabbits were separated into
groups of 100 individuals, placed in 10-ml beaker and im-
mersed in 5 ml of the formulations for 5 min, with each group
corresponding to a treatment. After immersion, the larvae
were dried on paper towels and divided into subgroups of
ten each, which were placed in labeled test tubes and sealed
with hydrophilic cotton, so that each tube corresponded to a
repetition. Therefore, ten repetitions were performed for each
treatment, with each repetition containing ten engorged larvae.
The mortality rates were assessed after 15 days, during which
period the tubes were kept in a climate-controlled chamber at
27 ± 1 °C and RH 80 ± 10%. The average mortality percent-
ages were calculated from the same equation used for the
unengorged larvae.
The same technique was used for the tests with engorged
nymphs, except each experimental unit contained five
nymphs, and each treatment was repeated ten times.
Data analysis
The Bioestat version 5.0 software was used for statistical anal-
ysis. The percent data of mortality were transformed (√arcsin
x) and analyzed by the Kruskal-Wallis and Student-Newman-
Keuls tests (p < 0.05). For the evaluation of pH averages,
Anova and Tukey’s tests were adopted.
Preliminary stability tests
The preliminary stability of the formulations was evaluated by
accelerated or short-term stability testing (Brasil 2004). In this
test routine, the formulations were stored for 15 days under
different stress conditions, to accelerate possible instability.
The samples were initially evaluated 24 h after preparation, by
sensory assessment, centrifugation and pH measurement, and
again after 15 days of stress. The storage conditions were (1)
high temperature (in an oven at 37 ± 2 °C), (2) low temperature
(in a refrigerator at 5 ± 2 °C), (3) room temperature (25 ± 2 °C),
with and without light exposure, and (4) freeze-thaw cycles
(24 h at 40 ± 2 °C and 24 h at 4 ± 2 °C, alternately).
These tests were only performed on the emulsion and
hydroalcoholic solution at concentrations of 2.5 and 5.0 mg/
mL, because these were the smallest concentrations that pre-
sented satisfactory acaricidal activity on more than one stage
of ticks tested. All the tests were performed in clear graduated
polypropylene tubes, properly sealed. The sensory evaluation
involved judging changes in color, odor, and appearance, after
subjecting the samples to stress.
The centrifugation was conducted on 10 mL of each sam-
ple, at 1509 g for 30 min at room temperature, followed by
observation of the presence or absence of macroscopic signs
of instability, such as creaming, coalescence, and flocculation.
The pH was measured in with a potentiometer after calibration
with buffer solutions at pH 4 and pH 7, on samples diluted
1:10 in recently distilled water, at room temperature
(25 ± 2 °C). Each sample was measured three times, and the
results presented correspond to the mean ± standard deviation
of the three readings. The sensory characteristics were ranked
in three categories: normal, without alteration (N); slightly
modified (SM); and intensely modified (IM) (Brasil 2004).
Results
In the tests with the emulsion formulations on unengorged
larvae, all the groups showed mortality near 100% starting at
the concentration of 0.75 mg/mL, in all cases, significantly
different than the control group. For engorged larvae, higher
thymol concentrations were necessary to reach relevant mor-
tality rates, which peaked at 95% for the concentration of
5.0 mg/mL. In turn, in the test with unengorged nymphs,
rising mortality rates were observed as the thymol concentra-
tion increased, with rates of 83.3% for the concentration of
2.5 mg/mL, rising to 100% with concentration of 20 mg/mL.
In the test with engorged nymphs, the average mortality was
86% at the concentration of 5.0 mg/mL (Table 1).
In the hydroalcoholic solution tests, the concentration of
2.5 mg/mL caused 88.1% mortality on unengorged larvae
and reached 98.1% at 5.0 mg/mL, and 100% at 10.0 and
20.0 mg/mL. The activity against engorged larvae was partial,
since the highest concentration tested (20 mg/mL) only caused
25.0% mortality, a value considered too low for effective tick
control (Brasil 1997). In the test with unengorged nymphs,
mortality of 91% was achieved at the concentration of
1.0 mg/mL, reaching 100% from 5.0 mg/mL onward, while
in the case of the engorged nymphs, the hydroalcoholic solu-
tion did not present relevant acaricidal activity, even at the
highest concentrations, attaining maximum mortality of only
18.3% with the highest concentration (20 mg/mL) (Table 2).
When submitting the O/W emulsion and hydroalcoholic
solution at the concentrations of 2.5 and 5.0 mg/mL to centri-
fugation in the initial stability triage, we noted that the emul-
sion at 5 mg/mL was intensely modified, with signs of coa-
lescence. This sample was, thus, excluded from the subse-
quent stability studies. The O/W emulsion at 2.5 mg/mL,
and the hydroalcoholic solution at both concentrations tested
 Parasitol Res

Table 1 Mortality rates

(mean ± SD) of immature stages Treatments Unengorged larvae Engorged larvae Unengorged nymphs Engorged nymphs
of Rhipicephalus sanguineus
sensu lato treated with different Control 0.8 ± 1.4a 3.0 ± 4.8ab 4.0 ± 8.4ª 4.0 ± 8.4a
ab
concentrations of thymol 0.5 mg/mL 73.2 ± 11.6 0.0 ± 0.0a 12.5 ± 16.3ab 3.3 ± 10.5a
incorporated in a O/W emulsion 0.75 mg/mL 94.2 ± 9.8bc 5.0 ± 7.1ab 41.7 ± 17.5b 2.0 ± 6.3a
under laboratory conditions
(27 ± 1 °C and RH 80 ± 10%) 1.0 mg/mL 100.0 ± 0.0c 16.0 ± 7.0bc 41.7 ± 15.7b 2.0 ± 6.3a
1.25 mg/mL 99.5 ± 1.3c 29.0 ± 14.5c 40.2 ± 25.2b 0.0 ± 0.0a
2.5 mg/mL 100.0 ± 0.0c 40.0 ± 15.6cd 83.3 ± 22.1c 32.0 ± 23.5b
5.0 mg/mL 100.0 ± 0.0c 95.0 ± 5.3de 86.7 ± 12.4c 86.0 ± 21.2bc
10.0 mg/mL 100.0 ± 0.0c 100.0 ± 0.0e 97.2 ± 8.3c 95.0 ± 9.3bc
20.0 mg/mL 100.0 ± 0.0c 100.0 ± 0.0e 100.0 ± 0.0c 100.0 ± 0.0c

Averages followed by equal letters in the same column do not differ statistically at a significance level of 5%.
Control = emulsion without thymol

did not show alterations after initial centrifugation, so they
were submitted to the storage challenge tests.
After exposure for 15 days to the different temperature and
lighting regimes, all the samples continued to have stable col-
or and characteristic thymol odor. The hydroalcoholic solution
was most stable in all the situations and at both concentrations.
The O/W emulsion at 2.5 mg/mL remained stable at room
temperature, with and without exposure to light, and when
kept under continuous refrigeration; but it showed signs of
instability (creaming) after the final centrifugation in the sam-
ples maintained in the oven and subjected to the freeze-thaw
cycle. The measurement of pH demonstrated a non-significant
variation (p < 0.05) between the initial values (1st day) and
final ones (15th day) for all the formulations tested, according
to the ANOVA and Tukey test. The results of the sensory
analysis, centrifugation, and pH measurement are shown in
Table 3.
Discussion
For many years synthetic substances were used with good
results to control ticks. However, due to their frequent
indiscriminate use and physicochemical characteristics, today
the occurrence of selection of resistant populations is becom-
ing more common. Additionally, these substances cause envi-
ronmental contamination and intoxication of animals and their
owners. Thus, it is necessary to find substances that are effec-
tive but have less impact on human and animal health and the
environment, besides imposing less selective pressure for the
resistance of ticks. Thymol has been studied for several years
as a bactericide, fungicide, acaricide, and insecticide
(Carvalho et al. 2003; Floris et al. 2004; Botelho et al. 2007;
Vasconcelos et al. 2014), and has been classified as a safe
substance by the US Environmental Protection Agency
(USEPA 1993). One of the reasons for this safety is that it
quickly dissipates in the environment, leaving low residue
levels (Hu and Coats 2008).
The results obtained in this study showed a stronger acar-
icidal effect of thymol on unengorged larvae in the tests with
the emulsion and hydroalcoholic solution compared to the
results reported by Daemon et al. (2009) and Daemon et al.
(2012). In the case of the engorged larvae, the emulsion was
more toxic. Indeed, the hydroalcoholic solution did not cause
relevant mortality rates, reaching levels lower than those ob-
tained when thymol was tested diluted in water + DMSO at

Table 2 Mortality rates

(mean ± SD) of immature stages Treatments Unengorged larvae Engorged larvae Unengorged nymphs Engorged nymphs
of Rhipicephalus sanguineus
sensu lato treated with different Control 0.1 ± 0.3a 7.5 ± 10.4ab 0.0 ± 0.0ª 0.0 ± 0.0a
ab
concentrations of thymol 0.5 mg/mL 3.1 ± 4.7 7.0 ± 8.2ab 4.5 ± 9.6ª 0.0 ± 0.0a
incorporated in a hydroalcoholic 0.75 mg/mL 7.7 ± 4.6bc 12.0 ± 10.3ac 5.0 ± 10.0ª 2.0 ± 6.3a
solution under laboratory
conditions (27 ± 1 °C and RH 1.0 mg/mL 6.5 ± 7.5abc 6.0 ± 8.4ab 91.0 ± 16.6b 0.0 ± 0.0a
80 ± 10%) 1.25 mg/mL 15.2 ± 3.5cd 8.0 ± 7.9a 92.0 ± 10.3b 2.0 ± 6.3a
2.5 mg/mL 88.1 ± 11.9de 0.0 ± 0.0b 96.0 ± 8.4b 0.0 ± 0.0a
5.0 mg/mL 98.1 ± 3.6e 8.0 ± 10.3ab 100.0 ± 0.0b 0.0 ± 0.0a
10.0 mg/mL 100.0 ± 0.0e 11.0 ± 12.9ac 100.0 ± 0.0b 0.0 ± 0.0a
20.0 mg/mL 100.0 ± 0.0e 25.0 ± 17.2c 100.0 ± 0.0b 18.3 ± 24.9ª

Averages followed by equal letters in the same column do not differ statiscally at a significance level of 5%.
Control = hydroalcoholic solution without thymol
Parasitol Res

Table 3 Results of the sensory analysis, centrifugation and pH measurement of the emulsion and hydroalcoholic solution, at concentrations of 2.5 and
5.0 mg/mL, in the accelerated stability test. The pH values are the mean ± SD of three consecutive measurements of each sample. N normal, SM slightly
modified, IM intensely modified

Storage period and conditions Emulsion Hydroalcoholic solution

2.5 mg/mL 5.0 mg/mL 2.5 mg/mL 5.0 mg/mL

Initial (24 h)
Sensory analysis N N N N
pH measurement 6.78 ± 0.05 – 6.12 ± 0.14 6.57 ± 0.03
Centrifugation N IM N N
After 15 days, at room temperature (25 ± 2 °C), with light exposure
Sensory analysis N – N N
pH measurement 6.35 ± 0.15 – 5.75 ± 0.07 6.16 ± 0.10
Centrifugation N – N N
After 15 days, at room temperature (25 ± 2 °C), without light exposure
Sensory analysis N – N N
pH measurement 6.26 ± 0.04 – 5.84 ± 0.08 5.86 ± 0.06
Centrifugation N – N N
After 15 days, in refrigerator (5 ± 2 °C)
Sensory analysis N – N N
pH measurement 6.67 ± 0.21 – 6.04 ± 0.11 5.96 ± 0.10
Centrifugation N – N N
After 15 days, in oven (37 ± 2 °C)
Sensory analysis N – N N
pH measurement 6.65 ± 0.05 – 5.99 ± 0.21 5.91 ± 0.05
Centrifugation SM – N N
After 15 days, under freeze-thaw cycles (40 ± 2 °C/4 ± 2 °C)
Sensory analysis N – N N
pH measurement 6.46 ± 0.10 – 5.80 ± 0.16 6.15 ± 0.03
Centrifugation SM – N N

1% (Daemon et al. 2009). For unengorged nymphs, both the
emulsion and solution attained similar mortality rates to those
obtained in a previous study using thymol dissolved in ethanol
(Senra et al. 2013). Finally, in the case of the engorged nymphs,
the groups treated with the emulsion suffered higher mortality at
the concentrations below 5.0 mg/ml, and as of this concentra-
tion the mortality rates were similar to those of the previous
study, where water + 1% DMSO was used. The hydroalcoholic
solution, in turn, did not reach relevant mortality rates: they
were below those reported by Monteiro et al. (2009).
The variations observed among the different stages tested
in this study can be attributed to the anatomic and physiolog-
ical difference of Ixodidae (hard ticks) at each stage (larva/
nymph) and phase (engorged/unengorged), and the chemical
properties of the formulation’s components.
Like all arthropods, ticks are covered with a tegument,
which besides body coverage and structural strength as the
exoskeleton, provide protection against water loss and act as
a source of reserve energy (Sonenshine 1991; Hackman and
Filshie 1982). The cuticle is the outermost part of the
tegument. Although most of this structure defined by
Hackman and Filshie (1982) as a Bheterogeneous, non-
cellular membrane^, is located on the external part of the tick
body, there is evidence that it extends inside the body at de-
termined points of communication with the tracheal system
and ducts of the dermal glands. Its density is not uniform over
the entire surface of the tick, so that there are thinner areas,
mainly found at articulation points, and thicker and harder
areas. The chemical composition of the cuticle is still not
totally resolved, but it is widely assumed to be composed
mainly of lipids, polyphenols, proteins, and chitin (Lees
1947; Hackman and Filshie 1982).
It is believed that the waxes present in the outermost layer
of the cuticle, called the epicuticle, are secreted by the dermal
glands, which are drained by pores to the surface (Sonenshine
1991). This secretion occurs more intensely during and imme-
diately after a blood meal or after exposure to light, heat, or
mechanical irritation. This is more evident in nymphs and
females (Lees 1947). Its function has been indicated as pro-
tection against desiccation (Sonenshine 1991).

Most water loss of ticks occurs through the cuticle (Lees
1947), and the lipid layer present in this structure plays an impor-
tant role in regulating this occurrence. This activity appears to
function by means of a physiological control, as demonstrated by
the regeneration of the lipid layer of the epicuticle of ticks after
production of artificial injuries by abrasion (Lees 1947) and by
changes in the proportion of lipids during the process of feeding
and ecdysis (Hackman and Filshie 1982). It has been confirmed
that various substances, such as detergents, when applied on the
surface of the cuticle of ticks can increase the transpiration rate,
leading to greater water loss (Lees 1947).
Larvae and nymphs of Ixodidae have been found to be
more susceptible to water loss than adults (Knüle and
Rudolph 1982). Besides the water loss that occurs through
the body tegument, additional water is lost by the gas ex-
change process during respiration (Sonenshine 1991). Adult
hard ticks as well as nymphs have a respiratory system com-
posed of spiracles and trachea. The larvae, however, do not
have these structures and perform gas exchange exclusively
through the cuticle (Hackman and Filshie 1982). Nymphs and
adults have a valve mechanism that opens and closes the spi-
racles, acting as another physiological control against water
loss (Needham and Teel 1991). The control of the opening and
closing of the spiracles has been related to the water balance
status of the animal, and according to Sonenshine (1991),
engorged ticks are unable to keep the respiratory system
closed, causing them to lose water faster.
In this study, in both formulations, we used glycerin, a
viscous substance with hygroscopic and moisturizing proper-
ties, for the purpose of keeping the formulation from drying
out. This component forms a hydrophilic film on the surfaces
where it is applied (Ribeiro 2006). The mortality rates found
in the tests with unengorged larvae suggest a possible delete-
rious effect caused by glycerin associated with thymol, possi-
bly resulting in increased water loss through the cuticle
(Ravindran et al. 2011), or even the formation of a film caus-
ing occlusion of the channels responsible for gas exchange,
since as mentioned above, at this stage, ticks do not have a
tracheal respiratory system. This effect was not observed in
the unengorged nymphs, and the mortality rates were similar
to those recorded in a previous study, where a hydroalcoholic
solution of 50% ethanol as the solvent was used (Senra et al.
2013). Since nymphs do not depend exclusively on the cuticle
for gas exchange and absorption of water (because they have a
tracheal system), the occlusive effects of the formulations on
the tegument might have been counterbalanced by its respira-
tory activity, so that the deleterious effect of the adjuvants
associated with thymol in the two formulations was attenuat-
ed. However, since no significant mortality occurred in the
control groups, the mere obstructive action of glycerin does
not explain the enhanced toxic effect of thymol. Thus, other
pharmacological and/or physicochemical hypotheses should
be investigated to clarify this enhancement.
Parasitol Res
The results of the tests with engorged larvae showed higher
mortality rates in the groups treated with the emulsion. In the
case of the hydroalcoholic solution, even at the highest con-
centrations tested, the mortality rate was not relevant. In a
previous study investigating thymol dissolved in water +1%
DMSO, relevant mortality rates (97%) were only attained
starting at the concentration of 15 mg/mL (Daemon et al.
2009). The increased toxicity in the treatment with the emul-
sion on engorged larvae can be related to the use of adjuvants
that promote permeation with lipophilic properties, such as
cetearyl alcohol and grape seed oil. Given the liposolubility
of thymol, we can suggest that the emulsion allowed stronger
interaction of that substance with the cuticle’s fatty layer, fa-
cilitating its entry in the organism, and also keeping it in sur-
face contact with the tick longer by the possibility of forming a
film over the tegument. In the tests of the emulsion on the
engorged nymphs, similar mortality rates were found to those
reported in a previous study using an aqueous solution of 1%
DMSO as solvent (Monteiro et al. 2009). On the other hand,
lower mortality rates were noted in the tests with engorged
nymphs and larvae treated with the hydroalcoholic solution.
To understand this phenomenon, it is important to consider the
higher production of waxes by the dermal glands that happens
in the engorged phases, as explained previously. It is possible
that the hydroalcoholic solution generated partial or inade-
quate solubilization of the waxes present on the cuticle’s sur-
face, making penetration of the thymol less successful. On the
other hand, the interaction of the lipophilic penetration agents
with the thymol and the fatty layer of the cuticle can explain
the fact the emulsion achieved satisfactory toxicity levels.
With respect to the stability studies, the hydroalcoholic
solution at concentrations of 2.5 and 5.0 mg/mL remained
stable in all the storage conditions tested. The emulsion at
2.5 mg/mL, in turn, showed signs of instability (creaming)
when subjected to high heat and freeze-thaw cycles.
However, according to Brasil (2004), small alterations in these
conditions are acceptable, frequent, and even expected.
Besides this, there was no significant variation in the pH
values which are suitable for application in the skin of dogs,
considering that the average pH of the skin of a healthy dog
varies between 5.86 and 6.45, with mean of 6.16 (Laurel,
2005). The emulsion at 5.0 mg/mL showed changes after the
initial centrifuging, with signs of coalescence, so it was not
tested under other conditions. Because this study is ground-
breaking regarding the incorporation of thymol as an acaricide
in formulations for topical use, the data presented here have
considerable value as support for new studies.
To better understand the action of the formulations tested
here, it will be necessary to conduct further studies to deter-
mine the exact mechanism by which thymol acts, as well as
the action of the moisturizing components used.
Accompanying investigations to adjust proportions of the ad-
juvants, and possibly the addition of other excipients, can
Parasitol Res
make the proposed formulations more stable, favoring a study
in the near future of their acaricidal effect in vivo, thus, con-
tributing to the validation of integrated strategies to control
R. sanguineus sensu lato.
Acknowledgements The authors thank the Conselho Nacional de
Desenvolvimento Científico e Tecnológico [CNPq (National Council
for Technological and Scientific Development)] and the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior [CAPES
(Coordination for the Improvement of Higher Education Personnel)],
Brazil, for the financial support of this study.
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