Efeito de óleos essenciais sobre Haemonchus contrortus

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Industrial Crops & Products
journal homepage: www.elsevier.com/locate/indcrop
Effects of Eucalyptus citriodora essential oil and its major component,
citronellal, on Haemonchus contortus isolates susceptible and resistant to
synthetic anthelmintics
José Vilemar de Araújo-Filhoa
, Wesley L.C. Ribeiroa,b
, Weibson P.P. Andréa
,
Géssica S. Cavalcantea
, Melissa de C.M. Guerraa
, Celli R. Munizc
, Iara T.F. Macedoa
,
Fernanda C.M. Rondona
, Claudia M.L. Bevilaquaa
, Lorena M.B. de Oliveiraa,⁎
a
Laboratório de Doenças Parasitárias, Programa de Pós-Graduação em Ciências Veterinárias, Universidade Estadual do Ceará, Fortaleza, Ceará, Brazil
b
Núcleo de Pesquisa e Desenvolvimento de Medicamentos, Universidade Federal do Ceará, Fortaleza, Ceará, Brazil
c
Empresa Brasileira de Pesquisa Agropecuária, Embrapa Agroindústria Tropical, Fortaleza, Ceará, Brazil
A R T I C L E I N F O
Keywords:
Anthelmintic
Eucalyptus citriodora
Citronellal
Inbred-susceptible Edinburgh
Kokstad
A B S T R A C T
This study aimed to evaluate the effects of Eucalyptus citriodora essential oil and citronellal on a Haemonchus
contortus isolate susceptible to anthelmintics, the Inbred-susceptible Edinburgh (ISE) and a resistant Kokstad iso-
late. The oil was analyzed by gas chromatography (GC). The products were evaluated using an egg hatching test
(EHT), a larval development test (LDT) and an adult worm motility test (AWMT). The effective concentrations to
inhibit 50% (EC50) of egg hatching and larval development and resistance ratios (RRs) were calculated. The
results were analyzed by analysis of variance and Tukey’s or Bonferroni’s test. The major constituent of E.
citriodora oil was citronellal (63.9%). According to EHT, EC50 values for the ISE isolate were 0.4 mg/ml and
0.3 mg/ml; for the Kokstad isolate, values were 0.5 mg/ml and 0.4 mg/ml for oil and citronellal, respectively.
LDT EC50 values were 2.9 mg/ml and 2.3 mg/ml for the ISE isolate and 3.2 mg/ml and 2.4 mg/ml for the
Kokstad isolate for oil and citronellal, respectively. In AWMT, 2 mg/ml of oil and citronellal completely inhibited
the motility of both the ISE and Kokstad isolates. RRs between the isolates ranged from 1 to 1.3. No significant
differences were observed between the isolates or treatment comparisons (P > 0.05). Therefore, E. citriodora oil
and citronellal both exhibited ovicidal and larvicidal effects and inhibited the motility of both H. contortus
isolates.
1. Introduction
Gastrointestinal nematode parasitism is one of the main limiting
factors of small ruminant production that endangers the well-being and
health of sheep and goats worldwide. Haemonchus contortus is con-
sidered one of the most prevalent and pathogenic parasites of small
ruminants in Brazil (Ashraf and Prichard, 2014; Ferreira et al., 2013).
The most commonly used method for controlling gastrointestinal
parasitism is the administration of synthetic anthelmintics. However,
the use of these drugs has promoted selection of resistant nematode
populations globally, and anthelmintic resistance is currently con-
sidered the main obstacle for parasite control (Kotze and Prichard,
2016; Sutherland and Leathwick, 2010). Accordingly, the search for
alternative control methods has become essential. Among these
alternatives, the use of secondary metabolites derived from plants has
received attention (André et al., 2016; Ribeiro et al., 2015; Torres-
Acosta and Hoste, 2008).
Many studies suggest that essential oils, which are complex mixtures
of volatile constituents produced by aromatic plants and used as de-
fenses against harmful agents, may have anthelmintic activity (Macedo
et al., 2011; Ribeiro et al., 2014). Promising results for inhibition of egg
hatching, larval development and adult worm motility test have been
obtained after incubation of essential oils with nematodes (Ferreira
et al., 2016; Macedo et al., 2015). Reduction in nematode egg excretion
and worm burden have also been recorded in goats and sheep after
administration of plant secondary metabolites (Macedo et al., 2011;
Ribeiro et al., 2015). Although essential oils can contain more than 60
compounds, two or three compounds are major constituents and
https://doi.org/10.1016/j.indcrop.2018.07.059
Received 14 May 2018; Received in revised form 19 July 2018; Accepted 24 July 2018
⁎
Corresponding author at: Programa de Pós-graduação em Ciências Veterinárias/FAVET/UECE, Av. Dr. Silas Munguba, 1700, Campus do Itaperi, CEP: 60.714-903,
Fortaleza, Ceará, Brazil.
E-mail address: lorena.mayana@uece.br (L.M.B. de Oliveira).
Industrial Crops & Products 124 (2018) 294–299
Available online 08 August 2018
0926-6690/ © 2018 Elsevier B.V. All rights reserved.
T

2. present in the highest concentrations. In general, the major constituents
and synergic interactions among these compounds determine the bio-
logical properties of essential oils (Bakkali et al., 2008; Ballhorn et al.,
2009).
Eucalyptus, belonging to the family Myrtaceae, is one of the most
cultivated trees in several countries, including Brazil. Among the spe-
cies of eucalyptus used for obtaining essential oils, Eucalyptus citriodora
is commonly used. E. citriodora essential oil is extracted from dry leaves,
and its major constituent is the monoterpenoid citronellal (60–80%)
(Hasegawa et al., 2008; Vitti and Brito, 2003). Studies have shown that
E. citriodora oil has multiple properties, including antioxidant (Singh
et al., 2012), antifungal (Brito et al., 2012), antibacterial (Cimanga
et al., 2002), anti-inflammatory and analgesic (Gbenou et al., 2013),
insecticidal (Maciel et al., 2010) and acaricidal (Clemente et al., 2010)
activities.
With regard to anthelmintic activity, E. citriodora essential oil has
exhibited action against H. contortus both in vitro and in vivo. However,
the level of resistance of this nematode to synthetic anthelmintics has
not been characterized (Macedo et al., 2011; Ribeiro et al., 2014). The
geographical origin of nematodes, as well as the resistance pattern of
isolates to synthetic anthelmintics, may influence the effect of plant
secondary metabolites (Chan-Pérez et al., 2016; Gaínza et al., 2016).
Thus, the present study was performed to evaluate the effect of E. ci-
triodora essential oil and citronellal, its major constituent, on different
life stages of H. contortus isolates susceptible and resistant to synthetic
anthelmintics.
2. Materials and methods
2.1. E. citriodora essential oil and citronellal
E. citriodora essential oil was purchased from Ferquima®
(São Paulo,
Brazil). The chemical composition of the oil was determined by gas
chromatography-mass spectrometry (GC–MS) using a GCMS-QP2010S
(Shimadzu®
, Japan). The following experimental conditions were em-
ployed: RTX-5 (30 m x 0.25 mm) capillary column; helium carrier gas;
injector temperature of 250 °C; detector temperature of 260 °C; column
temperature of 50–150 °C at 2.5 °C/min and then 150–250 °C at 25 °C/
min. The running time was 50 min. For mass spectrometry, the electron
impact was 70 eV. The components of E. citriodora oil were identified
according to their GC retention time, as expressed by Kovat’s index,
which was calculated using the Van den Dool and Kratz equation
(Adams, 2007). Additionally, test compound mass spectra were com-
pared to spectra from the National Institute for Standard Technology
computer database and published spectra. Quantification of E. citriodora
essential oil compounds was performed by the relative percentage of
peak areas of the chromatogram. Citronellal was purchased from
Sigma-Aldrich®
; according to the manufacturer, its purity determined
by GC was ≥ 95%.
2.2. H. contortus isolates
The Inbred-susceptible Edinburgh (ISE) isolate was used as a reference
for susceptibility because it is susceptible to all main classes of an-
thelmintics (Roos et al., 2004). The H. contortus Kokstad isolate was
used as a reference for resistance because it is resistant to benzimida-
zoles, levamisole and macrocyclic lactones (Fauvin et al., 2010; Neveu
et al., 2007). Both isolates were provided by the Institut National de la
Recherche Agronomique (INRA), Tours, France.
2.3. Recovery of H. contortus eggs, larvae and adults
Two sheep were housed in metabolic cages and dewormed with
5 mg/kg levamisole (Ripercol®), 0.2 mg/kg ivermectin (Ivomec®) and
2.5 mg/kg monepantel (Zolvix®). After total clearance of natural in-
fection, as confirmed by fecal egg counts (EPG) and coproculture, one
animal was monospecifically infected with 5000 H. contortus third-stage
larvae (L3) of the ISE isolate; the other was monospecifically infected
with 5000 H. contortus L3 of the Kokstad isolate. These animals were
used as a source of H. contortus eggs, larvae and adults for in vitro tests.
This study was approved by the Ethics Committee of the Universidade
Estadual do Ceará and registered under the number 2836026/2017.
2.4. In vitro tests
In vitro trials were used to evaluate the effects of E. citriodora es-
sential oil and citronellal against H. contortus, including the egg hatch
test (EHT), larval development test (LDT) and adult worm motility test
(AWMT). The susceptible and resistant isolates were evaluated in all
three tests. To increase solubility in aqueous media, essential oil and
citronellal solutions were prepared using 1% Tween®
80 (Vetec).
2.4.1. EHT
EHT was performed according to Coles et al. (1992). To recover H.
contortus eggs, feces were collected directly from the rectum of animals
harboring monospecific infection with H. contortus isolates and pro-
cessed according to the technique described by Hubert and Kerboeuf
(1992). Briefly, 250 μl of an egg suspension containing approximately
100 fresh eggs was incubated for 48 h at 25 °C with 250 μl of essential
oil or citronellal solution at different concentrations (0.125, 0.25, 0.5, 1
and 2 mg/ml). After this period, drops of Lugol®
were added to stop egg
hatching, and eggs and first-stage larvae (L1) were counted under a
microscope. This test was conducted with two controls: a negative
control with 1% Tween®
80 and a positive control with 0.025 mg/ml
thiabendazole. Three repetitions with five replicates for each treatment
and for each control were performed.
2.4.2. LDT
LDT was performed as described by Camurça-Vasconcelos et al.
(2007). An egg suspension was incubated for 24 h at 25 °C to obtain H.
contortus L1, and larval viability was evaluated. Next, 500 μl of larval
suspension containing approximately 250 L1 and 500 μl of essential oil
or citronellal solution at different concentrations (0.5, 1, 2, 4 and 8 mg/
ml) was incubated with 1 g of nematode-free feces for six days at room
temperature (27 °C). After this period, L3 were recovered according to
the methods described by Roberts and O’Sullivan (1950), and drops of
Lugol®
were added. The L3 were counted under a light microscope. The
following controls were employed: 1% Tween®
80 (negative) and
0.008 mg/ml ivermectin (positive). Three repetitions with five re-
plicates for each treatment and for each control were performed.
2.4.3. AWMT
AWMT was performed based on the methodology described by
Hounzangbe-Adote et al. (2005). Adult worms were collected from both
experimentally infected sheep. Immediately after euthanasia, their
abomasa were removed, opened and placed at 37 °C in saline solution.
Mobile adult females were rapidly collected and placed into 24-well
plates at a ratio of 3 worms per well in addition to 1 ml of phosphate-
buffered saline (PBS) enriched with 4% penicillin/streptomycin (Sigma-
Aldrich®
) at 37 °C. After 1 h of incubation (37 °C, 5% carbon dioxide),
1 ml of E. citriodora essential oil or citronellal at 2 mg/ml was added to
the worms. PBS with 4% penicillin/streptomycin and 100 μg/ml iver-
mectin were used as negative and positive controls, respectively. After
3 h, 6 h and 12 h of incubation, the motility and survival of adult worms
were observed under an inverted microscope. Eight replicates for each
treatment and for each control were performed.
2.4.4. Scanning electron microscopy (SEM)
The nematodes from AWMT, exposed for 12 h to 2 mg/ml of oil and
citronellal, and the negative control were fixed in Karnovsky solution
and rinsed three times in PBS. After this, the samples were fixed in 1%
osmium tetroxide solution, washed three times in distilled water and
J.V. de Araújo-Filho et al. Industrial Crops & Products 124 (2018) 294–299
295

3. dehydrated in a graded ethanol series (70%, 80%, 90% and 100%). The
next step was critical point drying in liquid carbon dioxide using an
EMS 850 critical point drying apparatus. Finally, the nematodes were
placed on metal stubs, coated with a layer of gold and visualized in a
Zeiss 940A microscope at an accelerating voltage of 15 kV.
2.5. Statistical analysis
The efficacy of each treatment in EHT was determined using the
formula (number of eggs/number of eggs + number of L1) ×100. In
LDT, the following formula was used: efficacy = [(number of L3 in the
negative control–number of L3 in the treated group)/number of L3 in
the negative control] ×100. Adult worm motility inhibition according
to AWMT was calculated as the number of motionless worms/total
number of worms per well ×100.
The results of EHT, LDT and AWMT were analyzed by analysis of
variance (ANOVA) using the software Graph Pad Prism®
5.0. One-way
ANOVA followed by Tukey’s test was used to compare data regarding
the same treatment and the same H. contortus isolate (P < 0.05). Two-
way ANOVA followed by Bonferroni’s test was performed to compare
data between treatments and nematode isolates (P < 0.05). The results
are expressed as the mean efficacy percentage of egg hatching, larval
development or adult motility inhibition ± standard deviation.
The effective concentration to inhibit 50% (EC50) of egg hatching
and the EC50 of larval development were determined by linear re-
gression using the SPSS 17.0 program. EC50 s were analyzed by two-
way ANOVA followed by comparison with Bonferroni’s test
(P < 0.05). The resistance ratio (RR) was calculated with the following
formula: RR = EC50 of the resistant isolate/EC50 of the susceptible
isolate (Sangster and Dobson, 2002).
3. Results
The chemical composition of E. citriodora essential oil is shown in
Table 1. GC–MS analysis confirmed that citronellal (63.9%) is the major
constituent of the essential oil. The presence of other constituents was
also revealed, including neo-isopulegol (8.2%), citronellol (5.2%) and
iso-isopulegol (4.7%).
The effects of E. citriodora essential oil and citronellal in EHT against
isolates of H. contortus susceptible and resistant to synthetic anthel-
mintics are shown in Table 2. The oil and citronellal exhibited ovicidal
activity at all tested concentrations, and the effect was dose dependent.
For the susceptible isolate, thiabendazole had a better effect than the
highest concentration of oil tested. In contrast, the highest concentra-
tions of oil and citronellal were more effective against the resistant
isolate than was the positive control. The EC50s of the oil were 0.4 and
0.5 mg/ml for the ISE and Kokstad isolates, respectively, and the EC50s
of citronellal in this assay were 0.3 and 0.4 mg/ml, respectively
(P > 0.05).
The effects of E. citriodora essential oil and citronellal in LDT against
the susceptible and resistant isolates of H. contortus are summarized in
Table 3. The oil and citronellal displayed larvicidal activity at all tested
concentrations, with the effect being dose dependent. Ivermectin had a
better effect against both isolates than did the highest concentration of
oil tested. The highest concentration of citronellal had the same effect
as the positive control against the ISE and Kokstad isolates. The EC50 s
of the oil were 2.9 and 3.2 mg/ml for the ISE and Kokstad isolates, re-
spectively. The EC50 s of citronellal in this assay were 2.3 and 2.4 mg/
ml for the susceptible and resistant isolates, respectively (P > 0.05).
In AWMT, 2 mg/ml of E. citriodora essential oil and citronellal
completely inhibited the motility of both H. contortus isolates at 3 h
post-exposure. The oil and citronellal had the same efficacy as iver-
mectin (100% inhibition at 12 h) (P > 0.05) against the susceptible
isolate. Regarding the resistant isolate, the oil and citronellal were more
effective than was the positive control (75% inhibition) (P < 0.05).
These data are presented in Table 4.
The EC50s of the oil and citronellal and the RRs obtained for both H.
contortus isolates in EHT and LDT are provided in Table 5. In general,
the EC50s of the E. citriodora essential oil and citronellal were slightly
lower for the ISE isolate than the Kokstad isolate. However, no sig-
nificant differences were observed between the isolates or between the
E. citriodora essential oil and citronellal (P > 0.05).
The parasites exposed to E. citriodora essential oil and citronellal
exhibited no significant cuticular changes compared with worms
treated with the negative control (Supplementary material).
4. Discussion
The use of natural products derived from plants as alternatives for
controlling gastrointestinal nematodes of small ruminants has been
extensively researched. Among compounds evaluated for anthelmintic
efficacy, extracts, essential oils and their constituents are noteworthy
(André et al., 2016; Cavalcante et al., 2016; Ribeiro et al., 2018).
In the present study, the anthelmintic potential of E. citriodora es-
sential oil and its major component, the monoterpenoid citronellal, was
evaluated using eggs, larvae and adults H. contortus isolates susceptible
and resistant to synthetic anthelmintics. Among the main disadvantages
that may limit the use of natural products as anthelminthic therapies
are the high qualitative and quantitative variations in bioactive com-
position. This variation is a result of the action of different factors on
the plant, such as the soil, climate, season, and phenological stage, as
well as the part of the plant used, plant chemotype and extraction
methodology employed (Elechosa et al., 2017; Matias et al., 2016;
Vaiciulyte et al., 2017).
Our chromatographic analysis of the E. citriodora essential oil used
in this study revealed citronellal as the major constituent (63.9%). This
result is in accordance with other studies in which citronellal was found
to be the major constituent of the E. citriodora essential oil. Nonetheless,
citronellal has been found in variable concentrations, such as 53.1%
(Hussein et al., 2017), 60.7% (Singh et al., 2012), 67.5% (Ribeiro et al.,
2014), 71.8% (Macedo et al., 2011) and 86.8% (Ribeiro et al., 2018).
The biological activity of essential oils is often attributed to the
major compound present. Thus, in addition to essential oils, isolated
bioactive constituents have also been evaluated for their anthelmintic
properties (Bakkali et al., 2008). Oils and isolated compounds studies to
date include Croton zehntneri and anethole (Camurça-Vasconcelos et al.,
2007), Cymbopogon citratus and citral (Macedo et al., 2015) and Thymus
vulgaris and thymol (Ferreira et al., 2016). Conversely, evaluation of the
Table 1
Composition of Eucalyptus citriodora essential oil as determined by gas chro-
matography-mass spectrometry (GC–MS).
Constituents KIlit KIexp Percentage (%)
Alpha-pinene 942 940 0.46
Beta-pinene 981 980 0.87
Limonene 1030 1029 0.34
Eucalyptol 1033 1032 1.67
Bergamal 1052 1053 0.30
Linalool 1098 1099 0.34
Rose Oxide 1109 1111 0.27
Neo-isopulegol 1147 1149 8.23
Citronellal 1154 1157 63.94
Iso-isopulegol 1159 1162 4.72
Neoiso-isopulegol 1170 1173 0.39
Citronellol 1224 1228 5.24
Menthol < 8-hydroxy-neo > 1327 1333 0.59
Cytronellyl Acetate 1339 1346 3.27
Beta-caryophyllene 1408 1417 0.63
Total identified – – 91.26
KIlit: Kovats Index found in the literature; KIexp: Kovats Index for the experi-
ment.
The values in bold highlight the chemical constituents found in higher per-
centages in the essential oil.
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296

4. anthelmintic activity of citronellal has not been described thus far.
Another factor that can cause variation in the anthelmintic activity
of plant-derived compounds is the heterogeneity of H. contortus isolates.
This variation was initially described by Calderón-Quintal et al. (2010),
who evaluated the ability of four tannin-rich plant (Acacia pennatula,
Leucaena leucocephala, Piscidia piscipula and Lysiloma latisiliquum) acet-
one:water extracts to inhibit larval migration in three isolates of H.
contortus from Mexico. The evaluated isolates showed different re-
sponses to the treatments that were related to variation in their sus-
ceptibility to the tannins present in the extracts.
The anthelmintic potential of E. citriodora essential oil has been
described previously. Macedo et al. (2011) and Ribeiro et al. (2014)
obtained 98.8% and 97.1% egg hatching inhibition at concentrations of
5.3 and 4 mg/ml, respectively. These data differ from the results ob-
served in the present study: we obtained similar ovicidal effects of
96.46% (ISE isolate) and 97.15% (Kokstad isolate) using a lower con-
centration of the essential oil (2 mg/ml). In LDT, the efficacy of the
essential oil at a concentration of 8 mg/ml against H. contortus ISE and
Kokstad isolate larval development was 93.78% and 95.12%,
respectively. These values were slightly lower than those found by
Macedo et al. (2011) and Ribeiro et al. (2014), who obtained efficacies
of 99.71% and 99.7% at concentrations of 10.6 and 8 mg/ml, respec-
tively. The observed variation in the in vitro activity of the E. citriodora
essential oil between this study and the previously mentioned studies
may be related to two main factors: variation in the chemical con-
stitution of the oil and genetic variation of the isolates.
Although citronellal was found to be the major constituent of the
three E. citriodora essential oils described, the quantitative differences
in this monoterpenoid (63.9%, 67.5% and 71.77%) may be a source of
variation. Moreover, the interactions between other constituents,
whether they have synergistic or antagonistic actions, must also be
taken into account (Katiki et al., 2017). For H. contortus isolates,
Macedo et al. (2011) and Ribeiro et al. (2014) used an isolate from
northeast Brazil, whereas the ISE and Kokstad isolates we used were
original isolates from the United Kingdom and South Africa, respec-
tively (Fauvin et al., 2010; Roos et al., 2004; Neveu et al., 2007).
In the present study, slight differences were observed in the ovicidal
and larvicidal activities of E. citriodora essential oil and citronellal
Table 2
Mean efficacy (percentage ± standard deviation) of Eucalyptus citriodora essential oil and citronellal on egg hatching from susceptible (ISE) and resistant (Kokstad)
isolates of Haemonchus contortus.
Concentrations E. citriodora Citronellal
ISE Kokstad ISE Kokstad
2 mg/ml 96.46 ± 2.17Aa
97.15 ± 1.20Aa
99.36 ± 0.76Aa
99.35 ± 0.75Aa
1 mg/ml 73.96 ± 3.77Ba
69.91 ± 3.22Bb
84.49 ± 3.95Bc
82.04 ± 4.58Ba
0.5 mg/ml 55.48 ± 3.03Ca
39.2 ± 3.13Cb
70.34 ± 4.03Cc
68.32 ± 2.67Cc
0.25 mg/ml 30.07 ± 3.66Da
25.16 ± 2.54Db
33.0 ± 4.63Da
32.58 ± 4.97Da
0.125 mg/ml 10.82 ± 1.90Ea
10.73 ± 2.68Ea
16.13 ± 4.24Eb
11.37 ± 4.17Ea
Tween 80 (1%) 4.08 ± 1.38Fa
4.15 ± 1.27Fa
3.92 ± 1.01Fa
4.7 ± 0.75Fa
Thiabendazole (0.025 mg/ml) 100 ± 0.00Ga
91.74 ± 4.07Gb
99.96 ± 0.16Aa
93.1 ± 3.35Gb
Values represent averages ± standard deviations for three repetitions with five replicates for each treatment and for each control.
Capital letters compare the mean in the columns and lowercase letters compare mean in the rows. Different letters indicate significantly different values (P < 0.05).
Table 3
Mean efficacy (percentage ± standard deviation) of Eucalyptus citriodora essential oil and citronellal on larval development from susceptible (ISE) and resistant
(Kokstad) isolates of Haemonchus contortus.
Concentrations E. citriodora Citronellal
ISE Kokstad ISE Kokstad
8 mg/ml 93.78 ± 2.79Aa
95.12 ± 2.29Aa
98.35 ± 1.27Ab
98.28 ± 1.51Ab
4 mg/ml 57.19 ± 4.24Ba
51.37 ± 4.02Bb
69.85 ± 4.27Bc
68.57 ± 4.97Bc
2 mg/ml 30.75 ± 3.92Ca
21.74 ± 4.12Cb
36.99 ± 3.72Cc
34.54 ± 3.56Cc
1 mg/ml 10.53 ± 2.91Da
9.8 ± 3.49Da
13.37 ± 3.65Da
17.05 ± 4.08Db
0.5 mg/ml 4.72 ± 1.92Ea
4.46 ± 2.00Ea
6.55 ± 2.39Ea
5.49 ± 1.94Ea
Tween 80 (1%) 0.92 ± 1.34Fa
1.07 ± 1.52Fa
1.04 ± 1.71Fa
1.17 ± 1.91Fa
Ivermectin (0.008 mg/ml) 100 ± 0.00 Ga
99.23 ± 1.12Ga
100 ± 0.00 Aa
98.97 ± 1.40Aa
Values represent averages ± standard deviations for three repetitions with five replicates for each treatment and for each control.
Capital letters compare the mean in the columns and lowercase letters compare mean in the rows. Different letters indicate significantly different values (P < 0.05).
Table 4
Mean efficacy (percentage ± standard deviation) of Eucalyptus citriodora essential oil and citronellal on worm motility of susceptible (ISE) and resistant (Kokstad)
isolates of Haemonchus contortus.
Treatments ISE
Exposure time (hours)
Kokstad
Exposure time (hours)
3 h 6 h 12 h 3 h 6 h 12 h
E. citriodora (2 mg/ml) 100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
Citronellal (2 mg/ml) 100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
100 ± 0.00Aa
Ivermectin (0.10 mg/ml) 50 ± 17.82Ba
100 ± 0.00 Ab
100 ± 0.00Ab
16.67 ± 25.20 Bc
66.67 ± 30.86Bd
75.00 ± 23.57Bb
PBS 0.00 ± 0.00 Ca
0.00 ± 0.00 Ba
0 ± 0.00Ba
0.00 ± 0.00 Ba
0.00 ± 0.00Ca
0.00 ± 0.00 Ca
Values represent averages ± standard deviations for eight replicates for each treatment and for each control.
Capital letters compare the mean in the columns and lowercase letters compare mean in the rows. Different letters indicate significantly different values (P < 0.05).
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297

5. against the ISE and Kokstad isolates, suggesting that the pharmacody-
namics of the essential oil and citronellal differ from those of benzi-
midazoles (binding to β-tubulin), levamisole (nicotinic receptors for
acetylcholine agonist) and macrocyclic lactones (chloride channel
agonist), as these treatments are effective against both isolates, in-
cluding the resistant one (Kohler, 2001; Mccavera et al., 2009; Moreno-
Guzmán et al., 1998).
In AWMT, 2 mg/ml of E. citriodora essential oil and citronellal were
able to inhibit 100% of ISE and Kokstad isolate motility at all evaluated
times. This finding suggests that both products may be substantially
absorbed by the parasite’s tegument, perhaps due to the lipophilicity
and low molecular weight of citronellal (154.25 g/mol). However, it is
not possible to predict how these natural compounds may act to pro-
mote paralysis in H. contortus (Bakkali et al., 2008; Lenardão et al.,
2007).
Chan-Pérez et al. (2016) reported high RR values when evaluating
the ovicidal activity of acetone:water extracts of Acacia pennatula and
Onobrychis viciifolia, which are plants containing polyphenols, on 10
isolates of H. contortus from different geographic origins (Mexico,
France, Australia, South Africa and the United States). The RRs varied
from 2.0 to 6.4 (A. pennatula) and 3.7 to 45.7 (O. viciifolia), illustrating
the influence of the isolate on the anthelmintic activity of natural
products. Moreover, the RR values were approximately 1 (1–1.3), likely
due to differences in the types of plant secondary metabolites, essential
oils and isolated monoterpenoids as well as the smaller number of H.
contortus isolates evaluated.
The absence of considerable changes in the cuticle of H. contortus
isolates exposed to the E. citriodora essential oil and citronellal, as ob-
served by SEM, indicates that induction of cuticle damage is not the
mode of action of these products. Because both the oil and citronellal
were able to inhibit the motility of H. contortus, it is possible that their
mode of action is related to interactions with internal structures of the
parasite, which may result in physiological disorders and lead to death
(Brunet et al., 2011).
5. Conclusion
Eucalyptus citriodora essential oil and its major compound, ci-
tronellal, affected different life stages of H. contortus. It was possible to
observe a discrete influence of the isolates used on the efficacy of the
evaluated compounds. Further studies involving a greater number of H.
contortus isolates and assessing interactions between the constituents of
the essential oils and evaluating the probable mechanisms of action of
these natural products are of great importance to allow the best per-
formance of these products as alternative anthelmintics.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Acknowledgments
We would like to thank Dr. Jacques Cabaret from INRA, Tours,
France, for providing the ISE and Kokstad isolates of H. contortus. The
authors also thank the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) (458011/2014-2) for their financial
support. Mr. Araújo-Filho received a master research scholarship from
Coordenação de Pessoal de Nível Superior (CAPES). Dr. Bevilaqua has a
researcher fellowship from CNPq (303018/2013-5).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.indcrop.2018.07.059.
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Table 5
Effective concentration to inhibit 50% (EC50) and confidence intervals ob-
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Eucalyptus citriodora essential oil and citronellal and the resistance ratios (RRs)
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Treatments EC50 (EHT) RR EC50 (LDT) RR
ISE Kokstad ISE Kokstad
E. citriodora
(Estimated
citronellalx
)
0.4 mg/
mlAa
(0.384 –
0.500)
0.3 mg/ml
0.5 mg/
mlAa
(0.341 –
0.819)
0.3 mg/ml
1.25 2.9 mg/
mlBb
(2.056 –
4.347)
1.8 mg/ml
3.2 mg/
mlBb
(1.863 –
6.893)
2.0 mg/ml
1.1
Citronellal 0.3 mg/
mlAa
(0.296 –
0.380)
0.4 mg/
mlAa
(0.321 –
0.410)
1.33 2.3 mg/
mlBb
(1.617 –
3.533)
2.4 mg/
mlBb
(1.646 –
3.528)
1
Capital letters compare the mean in the columns and lowercase letters compare
mean in the rows. Different letters indicate significantly different values
(P < 0.05).
x
Estimated values by the percentage of citronellal in the Eucalyptus citriodora
essential oil determined by gas chromatography-mass spectrometry (GC–MS).
J.V. de Araújo-Filho et al. Industrial Crops & Products 124 (2018) 294–299
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