Volume11,Number1(ene-jun2017)
ÓrganooficialdelaAsociaciónPeruanadeHelmintologíaeInvertebradosAfines(APHIA)
Lima-Perú
VersiónImpresa:ISSN2218-6425VersiónElectrónica:ISSN1995-1043
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ISSN Versión impresa 2218-6425 ISSN Versión Electrónica 1995-1043
Neotropical Helminthology, 2017, 11(1), jan-jun: 53-59.
ORIGINAL ARTICLE / ARTÍCULO ORIGINAL
IN VITRO EVALUATION OF OVICIDAL ACTIVITY OF PLANT EXTRACTS AGAINST
HAEMONCHUS CONTORTUS (TRICHOSTRONGYLIDAE)
EVALUACIÓN IN VITRO DE LA ACTIVIDAD OVICIDA DE EXTRACTOS DE PLANTAS
CONTRA HAEMONCHUS CONTORTUS (TRICHOSTRONGYLIDAE)
¹ Laboratório de Parasitologia, Fundação Universitária Vida Cristã – FUNVIC,
Faculdade de Pindamonhangaba, Pindamonhangaba, São Paulo, Brasil.
² Laboratório de Farmacognosia, Fundação Universitária Vida Cristã – FUNVIC, Faculdade de Pindamonhangaba,
Pindamonhangaba, São Paulo, Brasil.
3 Laboratório de Parasitologia, Universidade de Taubaté – UNITAU, Taubaté, São Paulo, Brasil.
FUNVIC – Faculdade de Pindamonhangaba
Via Radialista Percy Lacerda, 1000 Pinhão do Borba, Pindamonhangaba, São Paulo – Brasil. CEP:12441-245.
*profmatheuscoelho@gmail.com
Matheus Diniz Gonçalves Coelho¹;Ana Theresa da Silva Árcega¹; Luiza Ohashi Bonnett¹;
1 1
Lucas Tobias Rodrigues Maciel ; Fernanda Bueno Sant'Anna Pereira ; Gokithi Akisue² &
3
Francine Alves da Silva Coêlho
ABSTRACT
Sheep industry is a branch in livestock that has been growing over the years. However, the control of
gastrointestinal parasites is considered one of the greatest barriers of this practice. In this sense,
Haemonchus contortus (Rudolphi, 1803) is a hematophagus parasite which can cause severe anemia,
development of mumps, and death. It is known that the inappropriate use of allopathic medicines has
promoted the emergence of resistance by this parasite. Thus, the present study aimed to evaluate the
ovicidal activity of plant extracts against H. contortus. Haemonchus contortus eggs were obtained from
fecal samples of naturally infected sheep, and purified by modification of Sheather's method. The purified
eggs were submitted to six alcoholic plant extracts, at concentrations of 12.5 mg / mL, 25 mg / mL and 50
mg / mL. The plants which have been evaluated were Allamanda cathartica L., Musa sp, Nerium oleander
L., Mirabilis jalapa L., Carica papaya L. and Brugmansia suaveolens (Humb. & Bonpl. ex Willd.)
Bercht. After 168 h, 50 uL aliquots were analyzed for the presence of eggs turned into blastomere, embryo,
or larvae. It was observed that the alcoholic extracts of N. oleander and M. jalapa were the most
promising, since it prevented the outbreak of larvae in all tested concentrations, thus presenting a high
potential for composite products intended for grazing decontamination.
Neotropical Helminthology
53
Keywords: Environmental decontamination – Haemonchiasis – Plant extracts
Neotropical Helminthology, 2017, 11(1), jan-jun Coelho et al.
RESUMEN
Palabras clave: descontaminación ambiental – Extractos vegetales – Hemonquiasis
La industria ovina es una rama en el ganado que ha estado creciendo a través de los años, sin embargo, el
control de los parásitos gastrointestinales se considera una de las mayores barreras de esta práctica. En este
sentido, Haemonchus contortus (Rudolphi, 1803) es um parásito hematófago que puede causar anemia
severa, el desarrollo de paperas, y la muerte. Se sabe que el uso inapropiado de medicamentos alopáticos
ha promovido la aparición de resistencia por parte de este parásito. Así, el presente estudio tuvo como
objetivo evaluar la actividad ovicida de extractos de plantas contra H. contortus. Los huevos de H.
contortus se obtuvieron a partir de muestras fecales de ovejas naturalmente infectadas y se purificaron
mediante la modificación del método de Sheather. Los huevos purificados se sometieron a seis extractos
alcohólicos, a concentraciones de 12,5 mg / mL, 25 mg / mL y 50 mg / mL. Las plantas evaluadas fueron
Allamanda cathartica L., Musa sp, Nerium oleander L., Mirabilis jalapa L., Carica papaya L. y
Brugmansia suaveolens (Humb. & Bonpl. ex Willd.) Bercht. Después de 168 h, se analizaron alıcuotas de
50 μL para determinar la presencia de huevos transformados en blastómero, embriones o larvas. Se
observó que los extractos alcohólicos de N. oleander y M. jalapa fueron los más prometedores, ya que
evitó el brote de larvas en todas las concentraciones ensayadas, presentando así un alto potencial para
productos compuestos destinados a la descontaminación de pastoreo.
INTRODUCTION oxygen, humidity and temperature, the eggs
present in stool form a larva inside the egg in 24 h.
This larvae hatch and needs microorganisms to
feed. This is L1 larvae. If the environmental
conditions remain favorable, between 5 and 10
days, the larvae will suffer cuticle L1 changes, and
evolve to L2, and finally L3, which is the infective
form (O'Connor et al., 2006).
The frequent and inappropriate use of allopathic
medicines generates intense selection pressure,
leading to a selective advantage for tolerant
parasites, allowing them to multiply in future
generations, resulting in decreased efficacy of
allopathic medicines and promoting the emergence
of resistance to them (Sawleha et al., 2010;
Gilleard, 2013).
In this sense it is important to develop research
aimed at the search for new elimination alternatives
of this parasite, either through the treatment of
animals, whether through the pasture
decontamination. Thus, the aim of the present
study was to evaluate the ovicidal activity of plant
extracts known to have antiparasitic activity
against H. contortus.
The sheep industry is a branch in livestock that has
been growing over the years due to its high profits
and low cost of management, however, control of
gastrointestinal parasites has been considered one
of the greatest barriers of sheep breeding because
they cause significant losses in the herd income
(Silva et al., 2011).
There are several species of gastrointestinal
nematodes of sheep, but the most remarkable
species is Haemonchus contortus (Rudolphi,
1803), because in addition to high frequency of
infection, it is the most serious, since this is the only
Strongylidae that feeds directly from blood which
can result in hypoproteinemia, anemia with
development of submandibular swelling,
weakness or fatigue after exercise and diarrhea,
often leading to death, particularly to young
animals (Getachew et al., 2007; Prasad et al.,
2008).
The strongylids parasites have two stages in its
cycle: free-living stage and parasitic phase. In free-
living phase, under appropriate conditions of
54
55
were obtained from fecal samples collected
directly from the rectum of naturally infected sheep
of Santa Inês breed, and purified by modifying the
Sheather's method (flotation at 1,600 rpm / min
saturated sucrose d = 1.2 g / mL) (Jurasek et al.,
2010), followed by collection of 2 mL of the
supernatant and repeated washings with distilled
water (by centrifugation at 1600 rpm / 1 min).
The ovicidal tests were performed with standard
amounts of eggs immersed in the evaluated
dilutions and the activity was determined by
observing the proportion of viable eggs after 24,
48, 72 and 168 (7 days) h of exposure.
The results were statistically assessed (level of
significance = 5%) using nonparametric method
(Kruskal-Wallis) according to the normality of the
results obtained, followed by Dunn's test,
respectively. For that we used the BioEstat 5.0
software.
In the present research was considered effective
only extracts that inhibited 100% of egg hatching.
After 48 h of experimentation, it was observed 90%
of egg hatching in the control group and, among the
total extracts tested, the most effective were those
obtained from M. jalapa and N. oleander, since
they inhibited 100% off egg hatching at
concentrations of 25 mg/ mL and 50 mg/ mL as
shown in table 1.
Similarly, after 72 h, the two aforementioned plant
extracts remained the same percentage of egg
hatching inhibition in concentrations of 25mg/ mL
and 50mg/ mL and remained effective in this
regard, including 168 h after exposure. It should be
noted, however, that among the two most effective
vegetables, N. oleander also presented ovicidal
activity at a concentration of 12.5 mg/ mL at all
time intervals in which the assessments were made,
as shown in table 2.
Among the other extracts evaluated, the one
obtained from A. cathartica showed to be effective
in inhibiting egg hatching only at a concentration of
50mg/ mL thus being less promising than the two
Neotropical Helminthology, 2017, 11(1), jan-jun Ovicidal activity of plant extracts against Haemonchus
The plant selection criteria was based on scientific
evidence established in the literature, according to
which these extracts have presented ovicidal
activity and larvicide against nematodes as
Ancylostoma sp. (Santos et al., 2013) and H.
contortus (Oliveira et al., 2010; Pereira et al.,
2013). For confirmation of botanical identity,
vouchers were deposited in the herbarium SPF -
University of São Paulo, whose respective
numbers are quoted after the scientific name for
each species studied. Six plant species were
selected to carry out this research: Allamanda
cathartica L. (Apocynaceae) – G. Akisue 041,
Musa sp – (Musaceae) G. Akisue 061, Nerium
oleander L. (Apocynaceae) – G. Akisue 040,
Carica papaya L. (Caricaceae) – G. Akisue 062,
Mirabilis jalapa L. (Nyctaginaceae) – G. Akisue
045 and Brugmansia suaveolens (Solanacae)
Willd. - G. Akisue 030.
To obtain the extracts of A. cathartica, N. oleander,
M. jalapa and B. suaveolens, leaves of each plant
were processed. However, seeds were used of C.
papaya and the flower of Musa were used for
obtaining extracts of these vegetables.
The collected plant material was processed in the
laboratory of Pharmacognosy (LAFAPLAM),
Faculty de Pindamonhangaba. After drying in an
oven at 45 ° C and pulverization using an electric
coffee grinder (Cuisinart®), alcoholic extracts
were prepared using ethanol (absolute alcohol PA)
as liquid extractor, by Soxhlet method, according
to the norms of the Brazilian Pharmacopoeia
(Farmacopeia Brasileira, 2010). The obtained
extract was concentrated by rotary evaporation
apparatus for evaporation of the solvent to obtain
crude extract, which, in turn, was diluted in
distilled water to obtain different concentrations to
be analyzed.
The experiments were performed at the Laboratory
of Parasitology (LAPAM) from the Faculty de
Pindamonhangaba, in which were evaluated the
ovicidal activity of the above statements, which
were tested on three different dilutions (50 mg /
mL, 25 mg / mL and 12.5 mg / mL).
To perform the ovicidal test, H. contortus eggs
MATERIAL AND METHODS
RESULTS
56
mentioned above. Moreover, the extract that
showed less effectiveness was the one obtained
from C. papaya, particularly after 72 and 168 h of
treatment, at concentrations of 12.5 mg and 25 mg,
since there was a high survival of larvae which was
statistically similar to that observed in the control
group (p> 0.05).
Neotropical Helminthology, 2017, 11(1), jan-jun
Table 1. Ovicidal activity of alcoholics extracts after 24 and 48 h of exposure, against H. contortus. BE:
blastomered egg; EL: egg with larvae and L: larvae.
24 h 48 h
12.5mg/mL
25mg/mL
50mg/mL
12.5mg/mL
25mg/mL 50mg/mL
BE
EL
L
BE
EL
L
BE
EL
L
BE
EL
L
BE
EL
L BE EL L
A. cathartica 2
18
0
0
20
0
1
19
0
0
19
1
3
15
2 0 20 0*
B. suaveolens
6
14
0
7
13
0
0
20
0
2
17
1
3
13
4 4 13 3
C. papaya
9
11
0
4
16
0
5
15
0
3
5
12
5
5
10 3 17 0*
M. jalapa
3
17
0
1
19
0
0
20
0
3
16
1
1
19
0* 7 13 0*
Musa sp.
1
19
0
1
19
0
1
19
0
15
2
3
14
3
3 12 4 4
N. oleander 2 18 0 2 18 0 6 14 0 6 14 0 7 13 0* 5 15 0*
Control 3 17 0 3 17 0 3 17 0 0 2 18 0 2 18 0 2 18
* signicant difference (p<0,05) compared to other groups. Similarly, after 72 h, the two aforementioned plant extracts remained the same.
Table 2. Ovicidal activity of alcoholics extracts after 72 and 168 h of exposure, against H. contortus. BE:
blastomered egg; EL: egg with larvae and L: larvae.
72 h 168 h
12.5mg/mL 25mg/mL 50mg/mL 12.5mg/mL 25mg/mL 50mg/mL
BE
EL
L
BE
EL
L
BE
EL
L
BE
EL
L
BE
EL L BE EL L
A. cathartica
0
19
1
6
12
2
5
15
0
0
19
1
2
13 5 4 16 0
B. suaveolens 2 12 6 3 11 6 2 10 8 3 7 10 5 4 11 5 8 7
C. papaya
0
0
20a
0
0
20a
3
12
5
0
4
16a
0
4
16a0 16 4
M. jalapa
3
16
1
3
17
0
7
13
0
7
13
0
5
15 0 6 14 0
Musa sp.
1
19
0
1
19
0
1
19
0
0
10
10
2
5
13 1 19 0
N. oleander
3
17
0
7
13
0
4
16
0
7
13
0
12
8
0 11 9 0
Control 0 3 17a0 3 17a0 3 17 1 0 19a1 0 19a1 0 19
a- Similar letters imply a non-signicant difference (p>0.05).
DISCUSSION suaveolens, except the last, which showed no
ovicidal activity in this research.
It is noteworthy that, in the study by Santos et al.
(2013), all the extracts had not presented ovicidal
activity, which possibly justified the need for
evaluation of larvicidal activity by these authors.
On the other hand, in our study, the most promising
extracts have already demonstrated effectiveness
The results presented above agree in part with those
obtained by Santos et al. (2013) which evaluated
the activity of alcoholic extracts of 10 different
vegetables against Ancylostomidae larvae, and of
these, four of them showed more promising, to
know N.oleander, M. jalapa, A. cathartica and B.
Coelho et al.
57
in the inhibition of eggs hatching, thus showing a
greater potential to control hemonchosis over
hookworm.
This evidence also shows that the eggs of H.
contortus are more susceptible to the action of plant
extracts analyzed, compared to eggs of hookworm,
thus posing a result of significant importance,
since, as previously mentioned, one of the greatest
barriers of sheep breeding is related to resistance
that this parasite presents with regard to allopathic
drugs (Sawleha et al., 2010).
Rout et al. (2014) demonstrated antimicrobial and
antioxidant activity of N. oleander and also
hypothesized that such activities are due to the
presence of active principles such as flavovonoids,
terpenes, alkaloids and saponins.
The remarkable biological activity of N. oleander
also has already been demonstrated by other
researchers (Al-Jubouri & Al-Khan, 2008; El-
Akhal et al., 2015). Al-Jubouri & Al-Khan (2008)
evaluated the inhibitory activity of N. oleander
extract against the in vitro growth of Leishmania
tropica promastigotes and observed that 2.5 mg /
mL of leaf extract was able to inhibit 71.9% of
promastigote growth for 96 hours of culture.
According to the aforementioned authors, such
inhibition may be due to the presence of steroidal
glycosides, such as oleandrin and nirin.
El-Akhal et al. (2015) evaluated the larvicidal
activity of this plant against larvae of Culex pipiens
L., the transmitting agent of the virus that causes
West Nile infection. These authors demonstrated a
toxic effect of the hydroethanolic extract with
100% mortality in the minimum concentration of
160mg / mL.
According to these authors, the larvicidal activity
of the hydroethanolic extract of N. oleander may be
due to the major components, among them
flavonoids, sterols, terpenes, tripterpenes and
coumarins. In addition, N. oleander leaves contain
a fairly toxic mixture of cardiotoxic glycosides,
among which oleandrin, oleandrigenine, digoxin,
digitonin, digitoxigenin, nerizoside, neritaloside
and odoroside, which may be poisonous to
humans, animals, fish, birds and some insects.
However, phytochemical studies with regard to
active constituents and their biological activities
necessarily need to be delineated (El-Akhal et al.,
2015).
In turn, M. jalapa is a plant that has been used for
centuries to treat various disorders (Rozina, 2016),
but there is still little known about its antiparasitic
property. Zachariah et al. (2012) evaluated the
antiparasitic activity of methanolic extract of M.
jalapa against Pheretima phostuma (Vailliant,
1868) , which is a geohelminth that has been widely
used for the initial evaluation of anthelmintic
activity in vitro, due to its anatomical and
physiological similarity with human parasite
nematodes. These authors observed that the
methanolic extract of M. jalapa was able to cause
paralysis and death of adult worms of P. phostuma,
being considered potentially useful as
anthelmintic.
According with Zachariah et al. (2012) the
methanolic extracts of M. jalapa were potent as
anthelmintic probably because of flavonoids,
glycosides and tannins in dose-dependent manner
giving shortest time of paralysis and death with
80% w/v concentration.
The fact that three of the extracts evaluated have
not proven effective, does not eliminate the
possibility that these may be promising, since
despite not having presented ovicidal activity, they
could cause significant mortality when tested for
larvicidal activity, highlighting in this sense the
importance of future studies aiming to evaluate this
property. For example, despite not having
demonstrated ovicidal activity in this research,
Musa sp. was efficient against H. contortus in the
concentration of 75mg / mL, with efficacy of 98.5
to 100%, in the inhibition of larval development, in
another study conducted by Oliveira et al. (2010).
On the other hand, the fact that a plant extract not
show significant activity in vitro, does not preclude
the possibility that it may be potentially useful for
the control of hemonchosis in vivo. In this sense,
with regard to species of Carica gender, the
expressionless ovicidal activity of the ethanol
extract of C. papaya seeds found in this study
shows that theoretically it would not be useful for
grazing decontamination, however Pereira et al.
(2013) showed that administration of this same
extract to naturally infected goats, was able to
reduce the number of OPG (eggs per gram of feces)
Neotropical Helminthology, 2017, 11(1), jan-jun Ovicidal activity of plant extracts against Haemonchus
58
at 49, 23% and 72%, respectively, thus being
potentially useful to use as antiparasitic or in
addition to allopathic therapy (Vieira, 2008).
It can be concluded that alcoholic extracts of A.
cathartica, M. jalapa and N. oleander are of a high
potential for use as grazing decontamination, to
control hemoncose in the sheep herd, given the
property of inhibiting egg hatch of this parasite in
vitro, but further research needs to be designed to
check this property on a larger scale and to
determine possible environmental impacts of
direct use in soil using ecotoxicity tests, for
example.
Neotropical Helminthology, 2017, 11(1), jan-jun
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El-Akhal, F, Guemmouh, R, Zoubi YE & Lalami,
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Received December 2, 2016.
Accepted January 19, 2017.