Chenopodium ambrosioides L. essential oil and ethanol extract on control of canine Ancylostoma spp.

Abstract

The use of Chenopodium ambrosioides L. has shown to be promising in the management of gastrointestinal nematodes. The objective of this study was to quantitate the yield and characterize the chemical composition of the essential oil of C. ambrosioides, as well as to evaluate the in vitro effect of the ethanolic extract and the essential oil in L3 of Ancylostoma spp. and the in vivo effect(s) of the essential oil in dogs. The effects of the ethanol extract and essential oil on Ancylostoma spp. were evaluated in vitro by exposing larvae to the extract at concentrations ranging from 0.005 g mL-1 to 0.2 g mL-1 and to essential oil at concentrations of 50, 100, and 150 ?L mL-1. For the in vivo test, 26 healthy dogs, naturally infected by Ancylostoma spp., were divided into three groups: F1 - cookies were administered without active principle; F2 - herbal cookies containing 37.5 ?L g-1 essential oil of C. ambrosioides L.; F3 - cookies plus a commercial formulation containing febantel, pyrantel, praziquantel, and ivermectin. Complete blood counts and serum biochemistry for AST, ALT, AF, urea, creatinine, total protein, and albumin were performed. The yield of the essential oil was 0.3% m v-1, and its major components included ?-terpinene (1.24%), p-cymene (4.83%), and ascaridol Z (87%) and E (5.04%) isomers. The concentrations of C. ambrosioides L. ethanol extract used were ineffective against Ancylostoma spp. larvae. The essential oil at a concentration of 150 ?L mL-1 was effective against L3 larvae. In the in vivo study in dogs, the herbal cookies containing C. ambrosioides L essential oil reduced the number of eggs per gram of feces.

Full text

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Semina: Ciências Agrárias, Londrina, v. 38, n. 4, p. 1947-1954, jul./ago. 2017
Received: July 06, 2016 - Approved: Dec. 06, 2016
DOI: 10.5433/1679-0359.2017v38n4p1947
Chenopodium ambrosioides L. essential oil and ethanol extract on
control of canine Ancylostoma spp.
Extrato etanólico e óleo essencial de Chenopodium ambrosioides L.
no controle de Ancylostoma spp. de cães
Jessica Nascimento Moraes Monteiro1; Anderson Barros Archanjo2;
Gabriela Porrio Passos3; Adilson Vidal Costa4;
Lenir Cardoso Porrio5; Isabella Vilhena Freire Martins5*
Abstract
The use of Chenopodium ambrosioides L. has shown to be promising in the management of
gastrointestinal nematodes. The objective of this study was to quantitate the yield and characterize
the chemical composition of the essential oil of C. ambrosioides, as well as to evaluate the in vitro
effect of the ethanolic extract and the essential oil in L3 of Ancylostoma spp. and the in vivo effect(s)
of the essential oil in dogs. The effects of the ethanol extract and essential oil on Ancylostoma spp.
were evaluated in vitro by exposing larvae to the extract at concentrations ranging from 0.005 g mL-1
to 0.2 g mL-1 and to essential oil at concentrations of 50, 100, and 150 μL mL-1. For the in vivo test,
26 healthy dogs, naturally infected by Ancylostoma spp., were divided into three groups: F1 – cookies
were administered without active principle; F2 – herbal cookies containing 37.5 μL g-1 essential oil
of C. ambrosioides L.; F3 – cookies plus a commercial formulation containing febantel, pyrantel,
praziquantel, and ivermectin. Complete blood counts and serum biochemistry for AST, ALT, AF, urea,
creatinine, total protein, and albumin were performed. The yield of the essential oil was 0.3% m v-1,
and its major components included α-terpinene (1.24%), p-cymene (4.83%), and (Z)-ascaridole (87%)
and E (5.04%) isomers. The concentrations of C. ambrosioides L. ethanol extract used were ineffective
against Ancylostoma spp. larvae. The essential oil at a concentration of 150 μL mL-1 was effective
against L3 larvae. In the in vivo study in dogs, the herbal cookies containing C. ambrosioides L essential
oil reduced the number of eggs per gram of feces.
Key words: Essential oil. Helminths. Herb of Santa Maria. Medicinal plants.
Resumo
O uso de Chenopodium ambrosioides L. tem se mostrado promissor no controle de nematoides
gastrintestinais. Objetivou-se vericar o rendimento e a composição química do óleo essencial de C.
ambrosioides, assim como avaliar o efeito in vitro do extrato etanólico e do óleo essencial em L3 de
Ancylostoma spp., e o efeito in vivo do óleo essencial para cães. Para a atividade larvicida as larvas de
Ancylostoma spp. foram colocadas na presença do extrato etanólico nas concentrações de 0,005 g mL-1,
0,01 g mL-1, 0,02 g mL-1, 0,03 g mL-1, 0,04 g mL-1; 0,05 g mL-1, 0,06 g mL-1, 0,08 g mL-1, 0,1 g mL-1, 0,12
1 Discente, Programa de Pós-Graduação em Ciências Veterinárias, Universidade Federal do Espírito Santo, UFES, Alegre, ES,
Brasil. E-mail: jessicanmm@hotmail.com
2 Discente, Programa de Pós-Graduação em Biotecnologia, UFES, Vitória, ES, Brasil. E-mail: andersonarchanjo@gmail.com
3 Discente, Programa de Pós-Graduação em Ciência Animal nos Trópicos, Universidade Federal da Bahia, UFBA, Salvador, BA,
Brasil. E-mail: gporriopassos@gmail.com
4 Prof., Departamento de Química e Física, UFES, Alegre, ES, Brasil. E-mail: avcosta@hotmail.com
5 Profs., Departamento de Medicina Veterinária, UFES, ES, Brasil. E-mail: lenircp52@gmail.com; ivfmartins@gmail.com
* Author for correspondence

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Monteiro, J. N. M. et al.
g mL-1 e 0,2 g mL-1, do óleo essencial nas concentrações de 50 µL mL-1, 100 µL mL-1 e 150 µL mL-1. Para
o teste in vivo utilizaram-se 26 cães, hígidos, infectados naturalmente por Ancylostoma spp., divididos
em três grupos: F1 – foram administrados biscoitos sem princípio ativo; F2 – biscoitos toterápicos
com óleo essencial de C. ambrosioides L na concentração de 37,5 µL g-1; F3 biscoitos acrescido de
formulação comercial a base de febantel, pirantel, praziquantel e ivermectina. Foi realizado hemograma
completo e bioquímica sérica para AST, ALT, FA, ureia, creatinina, proteína total e albumina. Vericou-
se o rendimento de 0,3% m v-1 do óleo essencial e na sua composição química a presença de α-terpineno
(1,24 %), o p-cimeno (4,83 %) e ascaridol, nos seus isômeros Z (87%) e E (5,04 %), como componente
majoritário. O extrato etanólico de C. ambrosioides L., nas concentrações utilizadas, apresentou
insuciente atividade para o efeito larvicida. O óleo essencial na concentração de 150 µL mL-1 foi ecaz
contra L3. A formulação de biscoito toterápico com óleo essencial C. ambrosioides L reduziu o número
de ovos por grama de fezes dos cães.
Palavras-chave: Erva-de-santa-maria. Helmintos. Óleo essencial. Planta medicinal.
Introduction
Chenopodium ambrosioides L., popularly
known as Santa Maria herb and Paico, is a medicinal
plant of South America, which, for centuries, has
been popularly used for its medicinal properties
(GADANO et al., 2006).
The wide use of the Santa Maria herb is due to
the presence of high levels of ascaridole in the seeds,
leaves, and stem. It is mainly used for the treatment
of ascaridiasis, and has been demonstrated to be
effective in the control of Ascaridia sp. in Japanese
quails (VITA et al., 2015). Few authors have
reported the efcacy of the aqueous extract against
gastrointestinal nematodes in small ruminants
(SILVA, 2012) and hexane and dichloromethane
extracts against Toxocara canis larvae (REIS et al.,
2010).
According to Costa et al. (2002), the scientic
validation of phytochemical therapeutics is a
mandatory step for the correct use of medicinal
plants or their active compounds, and in vitro tests
allow for discovery of anthelminthic properties in
plant extracts, allowing the development of new
alternatives for the control of parasites.
In the control of nematode infections, drugs are
used, which can have unpleasant and unpalatable
avors, which hinder their administration in dogs.
To our knowledge, no study on the activity of C.
ambrosioides on Ancylostoma spp., as well as
its incorporation in dogcookies currently exists
in the literature. Sousa et al. conducted a survey
of anthelmintic activity of native plants of the
American continent in 2013, but did not report use
of C. ambrosioides on Ancylostoma spp.
The objective of this study was to determine the
yield and characterize the chemical composition of
the essential oil of C. ambrosioides, as well as to
evaluate the in vitro effects of the ethanolic extract
and the essential oil on L3 larvae of Ancylostoma
spp. and the in vivo effects of the essential oil in
dogs.
Materials and Methods
The aerial parts of C. ambrosioides were
collected in the morning from plants grown in a
greenhouse, located in the municipality of Alegre,
Espírito Santo State, Brazil, at coordinates of 20º 44
‘49’ ‘, latitude S, 41º27 ‘58’ ‘with a longitude W and
an altitude of 250 m. The exsiccate is deposited in
the Sector VIES Herbarium of Jerônimo Monteiro,
under registration VIES 21536.
To prepare the ethanolic extract, the leaves were
dried in a drying chamber at 40°C with dehumidier
until their weight remained constant. Dried leaves
were then ground to form powder with a grinder
(Walita HR2943 / 00), which was added to 20%
ethanol (weight/volume) (Vetec a 99.8% PA). This
mixture was kept at 27±2 ºC protected from light
for one week, then ltered, remacerated three more
times, and concentrated in a rotary evaporator at

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Chenopodium ambrosioides L. essential oil and ethanol extract on control of canine Ancylostoma spp.
40°C under reduced pressure to yield the ethanolic
extract which was 6% of starting leaf weight. Forty-
ve grams of extract were obtained, which was
stored at 4°C until use.
The essential oil was obtained from 100 g of
fresh leaves by hydrodistillation for 3 hours in a
Clevenger apparatus. After obtaining 300 mL of
hydrolysate (water + oil), the oil was extracted from
the aqueous phase using pentane (5x30 mL) as the
solvent. Anhydrous sodium sulfate was added to
the organic phase to remove water. The anhydrous
remainder was ltered, and pentane was removed
by rotary evaporator. The essential oil obtained was
conditioned in an amber glass bottle wrapped with
aluminum foil and stored at 4°C (CASTRO et al.,
2004; COSTA et al., 2013; PINHEIRO et al., 2013).
Identication of volatile components present
in the oil was performed by gas chromatography
coupled to a mass spectrometer (GC-MS), with
selective mass detector, model QP-PLUS-2010
(SHIMADZU). Chromatography was performed
with a fused silica capillary column with Rtx-5MS
stationary phase, 30 m long and 0.25 mm internal
diameter, using helium as the entrained gas. The
temperatures were 220 ºC in the injector and 300
ºC in the detector. The initial temperature of the
column was 60ºC, programmed to increase by 3 ºC
every minute, to a maximum temperature of 240
ºC (CASTRO et al., 2004; COSTA et al., 2013;
PINHEIRO et al., 2013).
Compounds were identied by comparing their
mass spectra with reference data from the equipment
database (NIST / EPA / NIH 08 Spectral Mass
Library), and with literature and Kovats indexes
(ADAMS, 2007).
The chemical constituents of the essential
oil were quantied by gas chromatography on a
SHIMADZU GC-2010 Plus instrument equipped
with a ame ionization detector (CG-DIC). The
entrainment gas used was nitrogen and the column
was a capillary column Rtx-5MS, 30 m long and
0.25 mm internal diameter. The injector and
detector temperatures were set at 240 and 250 °C,
respectively. The temperature setting in the oven
was the same as that used in GC-MS analyses.
The relative percentage of each component of the
essential oil was calculated by the ratio of the area
under its respective peaks over the total area under
all peaks in the sample (CASTRO et al., 2004).
This experiment was approved by the Ethics
Committee on Animal Experimentation of the
Faculdade de Filosoa, Ciências e Letras of Alegre,
ES, under number 0200018/2010.
The in vitro tests with ethanol extract and
essential oil were performed in duplicate for each
group by mixing 500 μL of extract or oil with 500
μL of the solution containing L3 larvae in a 1.5 mL
polypropylene tube. After 48 hours of treatment,
according to the method of Reis et al. (2010), 10 μL
of the solution was deposited on a glass slide and
motility of 30 larvae per eld was examined under
40x light microscopy. Larval activity was evaluated
according to the rubric of Reis et al. (2010), in which
0 = dead; 1 = immobile, but not dead; 2 = partial
body movement; 3 = slow whole body movement;
4 = intermediate whole body movement; 5 = rapid
whole body movement. Results are presented as
percentages of total represented by each possible
score. The larvae of Ancylostoma spp. were obtained
by fecal culture, adapted from Ueno and Gonçalves
(1998).
The following groups were evaluated in vitro:
Group A (negative control – distilled water); group
B1 (positive control – anthelmintic albendazole);
group B2 (positive control – praziquantel
combination, pyrantel pamoate and febantel in
liquid commercial form); group C (ethanolic extract
(EE) of C. ambrosioides at concentrations: C1:
0.005 g mL-1; C2: 0.01 g mL-1; C3: 0.02 g mL-1; C4:
0.03 g mL-1; C5: 0.04 g mL-1; C6: 0.05 g mL-1; C7:
0.06 g mL-1; C8: 0.08 g mL-1; C9: 0.1 g mL-1; C10:
0.12 g mL-1; C11: 0.2g mL-1); group D (essential oil
(EO) of C. ambrosioides at concentrations: D1: 50
μL mL-1; D2: 100 μL mL-1 and D3: 150 μL mL-1);

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Monteiro, J. N. M. et al.
and group E (solvent) E1: Propylene glycol and E2:
Polysorbate.
For the in vivo test, 26 healthy dogs, aged over
six months, weighing between 12 and 15 kg of live
weight, without distinguishing breed or sex, were
used, and not pregnant and housed in the Zoonosis
Control Center (CCZ) in the Municipality of Serra,
Espírito Santo. They were fed commercial feed and
water ad libitum. During the treatment, the dogs were
isolated in kennels with access to sunlight, away
from other animals. After clinical and laboratory
evaluation, otherwise healthy animals with natural
infection by Ancylostoma spp. were selected for the
study.
Cookies were prepared according to the
following formula: 1) water and salt cookie- 1.6
g (40%); 2) 20% pharmaceutical gelatin solution
– 0.4 g (10%); 3) vegetable fat – 0.2 g (5%); 4)
moist meat avored ration – 0.4 g (10%); 5) C.
ambrosioides essential oil – 150 μL; 6) dry ration
pellet – q.s.p. 4 g. After mixing, cookies were
molded and placed in an oven at 40°C for 4 hours.
The nal concentration of essential oil in each
cookie was 37.5 μL g-1. The animals were divided
into three experimental groups: Group F1: Dogs
treated with cookies without active principle (n = 9);
Group F2: Dogs treated with cookie with essential
oil of C. ambrosioides L. (n = 10); Group F3: Dogs
treated with cookie plus commercial product based
on febantel, pyrantel, praziquantel, and ivermectin
(n = 7). Each F1 and F2 animal received treatment
at the initial time (M1), one cookie per day for three
consecutive days, with repetition after 15 days. F3
animals received a single dose of the commercial
drug combination in cookie formulation at the dose
recommended by the manufacturer. This dose was
repeated after 15 days.
Blood samples were collected from the cephalic
vein for laboratory analysis. For blood counts, the
blood was collected at two time points: T1 – initial
time, immediately before the rst administration
of medication, and T2 – nal time, 21 days after
rst administration of the medication. This blood
was stored in 10% EDTA tubes and processed in
a Mindray BC- 2800Vet hematology autoanalyzer.
Differential counts of leukocytes were obtained
from blood smears stained with a Pantico rapid kit
(NewProv®).
Serum alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase
(FA), urea, creatinine, total protein, and albumin
were analyzed in a Bioplus Bio-200® biochemical
analyzer.
To verify the ability of the cookies to reduce
numbers of Ancylostoma spp. eggs, fecal samples
were obtained from the rectum of naturally infected
dogs. Eggs were detected by the method described
by Willis (1921), and quantied by otation and
counting of eggs per gram of feces (EPG) (SLOSS
et al., 1999). Fecal samples were collected at two
time points, immediately prior to administration of
cookies, and 21 days after the initial administration
of cookies. Percent reduction of EPG of feces
between initial and nal time points was calculated
for each group.
Values obtained were analyzed by the parametric
T test paired using the program BioEstat 5.0.
P values of 0.05 or less were taken to indicate
statistical signicance.
Results and Discussion
The essential oil was obtained from the aerial
tissues of C. ambrosioides in a yield of 0.3%
m v-1. Gas chromatographic analysis allowed
the quantication of 98.81% of the chemical
constituents present in this essential oil. The major
components were: (Z)–ascaridole (87%), (E)-
ascaridole (5.04%) and p-cymene (4.83%) in lower
percentages of α-terpinene (1.24%).
Singh et al. (2008) described α-terpinene as
the major compound of the essential oil of C.
ambrosioides. Tapondjou et al. (2002) reported
that p-cymene corresponded to 50% of the

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Chenopodium ambrosioides L. essential oil and ethanol extract on control of canine Ancylostoma spp.
chemical composition of the essential oil obtained
in Cameroon; in this work, only 4.83% was
p-cymene. The monoterpene ascaridole [major
components (isomer Z (87%) and isomer and E
(5.04%)] constituted only 0.7% of the essential
oil in Cameroon (CHEKEM et al., 2009, 2010).
Environmental stimuli can redirect metabolic routes,
causing biosynthesis of different compounds.
In the in vitro test, groups A, E, and C1 to C11
did not inhibit larval activity. All of these samples
scored 5. Groups B1 and B2, containing commercial
drugs, scored 2 and 3, respectively. Groups D1 and
D2 scored 4 and 3, respectively, and D3 scored zero
(Table 1).
Table 1. Results of in vitro testing of the anthelmintic activity of Chenopodium ambrosioides towards infective larvae
of Ancylostoma spp.
Groups Test Score % living
larvae
% immobile
larvae
A (negative control) distilled water 5 100,00 0,00
B1 (positive control) Albendazole 2 35,00 65,00
B2 (positive control) Praziquantel, pirantel pamoate and febantel 3 38,33 61,67
C1 a C11 (treatment EE) ethanolic extract 5 100,00 0,00
D1 (treatment EO) 50µL mL-1 essential oil 4 88,33 11,66
D2 (treatment EO) 100µL mL-1 essential oil 3 95,00 5,00
D3 (treatment EO) 150µL mL-1 essential oil 0 0,00 100,00
Groups C1 to C11 did not demonstrate toxicity in
vitro towards infective larvae (L3) of Ancylostoma
spp. All larvae of these groups were diluted with
propylene glycol and remained alive, demonstrating
that the solvent used to solubilize ethanol extracts
did not kill the larvae or inhibit their activity.
In in vitro tests of the essential oil, 88.33% of
the larvae treated with 50 μL mL-1 (D1) scored 4
and 11.66% of them scored 0. Ninety-ve percent
of larvae treated with 100 μL mL-1 essential oil
(D2) scored 3, and 5% scored 0. With the essential
oil at the concentration of 150 μL mL-1 (D3) the
larval activity was 100%. This essential oil contains
ascaridole, a monoterpene found in large amounts in
the leaves and fruits of Chenopodium ambrosioides
L. The plants in this study contained 87% (Z)-
ascaridole, which behaves as a potent anthelmintic
agent (CASTELLANOS, 2008).
In in vivo test subjects, a reduction in the red
cell count (p = 0.01), hematocrit (p = 0.01), and
CHCM (p = 0.001) were observed in group F1. A
reduction in CHCM was observed in group F2 (p =
0.04), while a reduction in lymphocytes (p = 0.03)
and increase in monocytes (p = 0.03) were observed
in group F3. Albumin was reduced in F1 serum (p
= 0.01). In group F2, increases were seen in serum
proteins (p = 0.005) and globulins (p = 0.001).
In group F3, albumin decreased (p = 0.007) and
globulins increased (p = 0.006).
In the in vivo test, the F1 group experienced a
signicant reduction in red blood cell parameters
(p = 0.01), hematocrit (p = 0.01), and CHCM (p =
0.001) associated with albumin reduction (p = 0.01),
suggests that the cause is the parasitic infection itself
during the drug test period. These results highlight
the importance of treatment because an infected
animal can develop anemia and hypoalbuminemia
in a short time.
Group F2 dogs, treated with 37.5 μL g-1 C.
ambrosioides essential oil, exhibited signicant
reduction in CHCM (p = 0.04) and increase in
serum proteins (p = 0.005) and globulins (p =

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Monteiro, J. N. M. et al.
0.001). Pereira et al. (2010) treated mice with a
hydroalcoholic extract of leaves of C. ambrosioides
at a dose of 5 mg kg-1 for 15 days and observed an
immune response with a signicant increase in the
number of hematopoietic cells.
The F3 group experienced a signicant reduction
in lymphocytes (p = 0.03) and an increase in
monocytes (p = 0.03) over the course of the study.
However, both pre- and post-treatment values were
within reference standards described by Weiss and
Wardrop (2010). Reduced albumin (p = 0.007) and
increased globulin (p = 0.006) concentrations were
also observed in this group.
We observed a 7.42% reduction in EPG in the F1
group, 82.14% reduction in the F2 group, and 100%
reduction in F3. The reduction in EPG in the F2 group
was statistically signicant (p <0.001). Although
treatment with essential oil of C. ambrosioides
did not yield negative results upon parasitological
examination, the reduction in EPG can be considered
satisfactory because the small amount of remaining
antigen is expected to stimulate the immune system.
Furthermore, according to the efcacy index for
adult parasites proposed by the World Association
for the Advancement of Veterinary Parasitology
(WAAVP), a product is considered effective when
the reduction in egg counts is greater than 90% and
moderately effective when this reduction is between
80% and 90% (COLES et al., 1992).
Conclusions
Based on the results of this study, we concluded
that the ethanolic extract of C. ambrosioides L.
did not possess anthelmintic efcacy towards L3
Ancylostoma spp. larvae. The essential oil of C.
ambrosioides L., at a concentration of 150 μL mL-1,
was effective in vitro against third-stage larvae of
Ancylostoma spp., and the herbal cookies containing
37.5 μL g-1 of C. ambrosioides L. essential oil
signicantly reduced the EPG in feces of dogs
naturally infected with Ancylostoma spp.
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