J Parasit Dis (Jan-Mar 2021) 45(1):252–257
https://doi.org/10.1007/s12639-020-01302-9
ORIGINAL ARTICLE
Ovicidal and toxicological effect of hydroalcoholic extracts
of Euphorbia milli var splendens, Synadenium carinatum Boiss
and Tagetes minuta L. against Ancylostoma spp.: In vitro study
Matheus Diniz Gonçalves Coêlho1,2 • Lucas Tobias Rodrigues Maciel1 •
Thaı́s de Fátima Kieko Ozaki1 • Maria Eduarda Godoi Silva1 • Lilian Saito Ormachea Bozo1 •
Yumi Ando Consoli1 • Fernanda Bueno Sant’Anna Pereira-Maciel1 •
Gokithi Akisue3 • Francine Alves da Silva-Coêlho4
Received: 10 July 2020 / Accepted: 27 October 2020 / Published online: 7 November 2020
Ó Indian Society for Parasitology 2020
Abstract Nematodes of the Ancylostomidae family consist of important parasitic species. The control of such
parasitosis represents an important challenge, given the
constant high rate of reinfection of some hosts, among
which the domestic dog, as well as the high environmental
contamination. Another factor that can have a negative
influence is the toxicity of the chemicals used for environmental decontamination, highlighting the need to design
research to identify new control strategies for this parasitosis, among which the use of plant extracts. Thus, the
objective of this research was to evaluate the toxicity and
ovicidal activity of hydroalcoholic extracts obtained by
percolation of three plants: Tagetes minuta L., Euphorbia
milli var splendens (Bojer ex Hook.) Ursch & Leandri and
Synadenium carinatum Boiss, against Ancylostoma spp. In
addition, these extracts were tested at different concentrations for toxicity against Artemia salina L. and Allium cepa
L. It was observed that the hydroalcoholic extract of all
plant species evaluated induced moderate ovicide activity
at all the tested concentration, with emphasis on E. milii,
which was the only plant species that presented
& Matheus Diniz Gonçalves Coêlho
profmatheuscoelho@gmail.com
1
Centro Universitário FUNVIC, Pindamonhangaba,
São Paulo, Brazil
2
Laboratório de Parasitologia e Malacologia, Centro
Universitário FUNVIC, Pindamonhangaba, São Paulo, Brazil
3
Laboratório de Farmacognosia e Plantas Medicinais, Centro
Universitário FUNVIC, Pindamonhangaba, São Paulo, Brazil
4
Laboratório de Parasitologia, Instituto Básico de Biociências
- IBB, Universidade de Taubaté - UNITAU, Taubaté,
São Paulo, Brazil
123
significantly low toxicity in the concentration of 12.5 lL/
mL when compared to the other species evaluated.
Keywords Plant extracts Environmental contamination
Ancylostoma spp Toxicity
Introduction
Hookworm are the most prevalent parasites in domestic
dog (Canis familiaris), and can also trigger pathological
processes in humans (Coêlho et al. 2013). The occurrence
of this parasite is related to environmental contamination,
and, in view of this, it becomes necessary to deploy control
measures, among which the treatment of infected hosts and
the decontamination of the environment, which brings as a
disadvantage the possibility of harmful effect to other
species of living beings, due to the toxicity of chemical
substances used for this purpose (Santos et al. 2013).
To get around this problem, the research of plant
extracts is promising, as certain species demonstrate high
biological activity and, consequently, can bring perspectives in the control of hookworm, being necessary, however, to consider the potential toxic risk to the environment,
despite knowing that the use of plant derivatives has a
lower risk of environmental impacts (Coêlho et al.
2018, 2019).
Some plant species have been considered promising for
the control of pathogenic or vector agents, among which
Tagetes minuta L., Synadenium carinatum Boiss (synonym: Euphorbia pereskiifolia) and Euphorbia milii var
splendens.
The nematicidal activity of T. minuta (Asteraceae), has
been demonstrated by several authors, who proved this
property against some phytonematoides of the
J Parasit Dis (Jan-Mar 2021) 45(1):252–257
253
Meloidogyne genus, which are associated with the root
system of plants, causing significant damage in agriculture
(Massuh et al. 2017).
Regarding S. carinatum (Euphorbiaceae) and Euphorbia
milii var splendens, these species have been identified as
plants with high potential for use against several targets,
among which, species of vector mollusks (Coêlho et al.
2019; Moreira et al. 2010) but little is yet described about
their antiparasitic properties against nematodes.
Otherwise, toxicity tests have been widely developed to
evaluate plant extracts, and among which are the tests
using Artemia salina L. and Allium cepa L. as bioindicators. The acute toxicity test on A. salina L. is internationally used due to its great reproductive potential, easy
acquisition on the market, easy hatching of specimens and
good reproducibility (Mendes et al. 2011). A. cepa L., can
be used to monitor the presence of toxic compounds, with
the most analyzed parameter being phytotoxicity, through
determination of root growth inhibition, root weight and
weight gain of the bulbs after exposure to a potentially
toxic substance (Fão et al. 2012).
Therefore, this study aimed to evaluate in vitro toxicity
of hydroalcoholic extracts of Euphorbia milli var splendens, Synadenium carinatum Boiss and Tagetes minuta L.,
and ovicidal activity against Ancylostoma spp. obtained
from naturally infected dogs.
The extracts were tested in triplicate and in three different
dilutions (100 lL/mL, 50 lL/mL and 12.5 lL/mL). Eggs
of Ancylostoma spp. obtained from naturally infected dogs
were purified by modifying the Sheathers method (centrifugal-flotation at 1600 rpm/5 min) (Neves et al. 2016).
After purifying the eggs, a suspension was prepared containing approximately 1500 eggs/mL, being aliquoted and
transferred to Falcon tubes with a capacity of 15 mL. The
extracts were added so that the eggs remained in contact
with them at room temperature.
The ovicidal activity was evaluated 24 h and 1 week
after the beginning of the experiment. For this purpose,
slides containing a 20 lL aliquot were made, with observation and evaluation of 30 eggs, using an optical microscope in the increments of 100 9 and 400 9 in order to
evaluate the effectiveness of the plant extracts with regard
to the evolution and viability of the eggs, being considered
viable those that were intact and with the presence of a
blastula or formed larvae. Eggs that were broken or with
embryo destruction were considered unfeasible. It should
be emphasized that, for the comparison and observation of
eggs, two negative control groups were evaluated, that is,
without the addition of extract, ensuring the effectiveness
of the test, as well as a positive control group, which was
exposed to Orto Benzil P-chloro phenol 70%, which
proved to be 100% effective in inhibiting Ancylostomidae
eggs (Santos et al. 2013; Suzuki et al. 2013).
Materials and methods
Artemia salina L. test
Plant material obtaining
The plant species used in the present study were collected
in urban areas in the municipality of Pindamonhangaba and
Piedade, both located in the state of São Paulo - Brazil, and
taken to the Laboratory of Pharmacognosy and Medicinal
Plants of the FUNVIC. Exsiccates were prepared and
deposited at the SPF herbarium of the University of São
Paulo. The material of each plant species was processed,
dried in an oven at 45 °C and pulverized, so that the
extracts could be continued.
About 10 nauplii were transferred to tubes containing 5 mL
of each plant extract, in the evaluated dilutions, using
saline as a solvent. This experiment was carried out in
triplicate.
It is worth mentioning that a positive control was performed, composed of three tubes, using 0.05 g potassium
dichromate solution (K2Cr2O7), dissolved in 50 mL of
saline water, as well as a negative control composed of
three test tubes containing 5 mL of saline water. The count
of dead nauplii was carried out after 24 h (Meyer et al.
1982).
Hydroalcoholic extract production
Test with Allium cepa L.
After spraying the plant material, the percolation technique
was performed, following the fractional percolation process C, as described by Furtado el al. (2013), obtaining a
dilution of 1:100.
To perform this test, all evaluated extracts were tested only
in the dilution of 12.5 lL/mL. Ten onions were used for
each extract, which were placed over sedimentation cups,
in order to constantly keep their root regions in contact
with them. A control group was also used, formed by 10
onions that remained in chalices containing only distilled
water. The samples were stored at room temperature,
remaining in contact with their respective solutions. Root
evaluation was performed 96 h after the start of the test,
Ovicidal activity test
The ovicidal activity assessment tests were carried out at
the FUNVIC Parasitology and Malacology Laboratory.
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determining quantity, growth and weight of the roots, and
weight gain of the bulbs.
Statistical analysis
In each experiment, the averages obtained from each group
were evaluated using analysis of variance (ANOVA test)
with Tukey’s test for confirmation, using the GraphPad
Prism 6.0 software as a tool.
Results and discussion
After carrying out the test using A. salina, there was no
toxicity of the hydroalcoholic extract of E. milii at the
concentration of 12.5 lL/mL, since the observed mortality
was not significantly higher than that observed by the
control group (p \ 0.005), as shown in Table 1.
These results were confirmed in the toxicity test using A.
cepa L. as a bioindicator, in which E. milli var. splendens
presented a low toxicity in two of the three evaluated criteria, since obtained results were statistically similar to
those obtained in the control group, namely: root numbers
and weight gain of the onion bulb (Figs. 1, 3, 4). In relation
to the other extracts tested, namely: T. minuta and S. carinatum, they showed toxicity both in the mortality test with
A. salina L. and in the test with A. cepa, particularly with
regard to S. carinatum which in this last test, showed a
higher toxicity (p \ 0.005) than what was observed in the
control group in all evaluated criteria (Figs. 1, 2, 3, 4).
It is known that for carrying out toxicity assay, the tests
recommended by the OECD - Organization for Economic
Cooperation and Development are considered standard
methods for the safety thesis and that the OECD guidelines
for testing of chemicals are a unique tool for asessing the
potential effects of chemicals on human health and environment (OECD 2012).
Although the tests used in the present research are not
part of the list of methods recommended by the OECD, the
toxicity tests using Allium cepa and the microcrustacean
Fig. 1 Evaluation of the toxicity of three plant extracts using the A.
cepa L. test in relation to the average number of roots. * = significant
difference in relation to the control group
Artemia salina have been used as tests of great potential
(Rajabi et al. 2015, Johari et al. 2019; Ciappina et al.
2017); and come upon the worldwide efforts to reduce,
refine and replace (3Rs) animals used in experiments, as
well as the BraCVAM (Brazilian Center for the Validation
of Alternative Methods) initiative, which aims to validate
alternative methods and encourage the incorporation of
new methodologies (Araújo et al. 2014).
The low toxicity of E. milli var. splendens observed in
the present study, against A. salina L. nauplii, agrees with
results obtained by Coêlho et al. (2018), who observed
their absence of mortality in low dilutions of latex of this
plant species, namely, 100 ppm, 50 ppm and 25 ppm.
The toxicity of T. minuta agrees with results presented
by Cunha et al. (2016), however, these authors evaluated
the toxicity against another target species, namely the silver catfish (Rhamdia quelem) and tested not the hydroalcoholic extract, but the essencial oil, which was obtained
Table 1 Toxicity test against A. salina using hydroalcoholic extracts, in concentrations of 100 lL/mL, 50 lL/mL and 12.5 lL/mL
Tagetes minuta L.
Synadenium carinatum Boiss
Euphorbia milli var splendens
Concentration
Mortality
Concentration
Mortality
Concentration
Mortality
100.0 lL/mL
30/30 (100%)*
100.0 lL/mL
30/30 (100%)*
100.0 lL/mL
30/30 (100%)*
50.0 lL/mL
30/30 (100%)*
50.0 lL/mL
28/30 (93.3%)*
50.0 lL/mL
26/30 (86.6%)*
12.5 lL/mL
27/30 (90%)*
12.5 lL/mL
16/30 (53.3%)*
12.5 lL/mL
4/30 (13.3%)
Negative control
0/30 (0%)
Negative control
0/30 (0%)
Negative control
0/30 (0%)
Positive control
30/30 (100%)
Positive control
30/30 (100%)
Positive control
30/30 (100%)
*Significant difference
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Fig. 2 Evaluation of the toxicity of three plant extracts using the A.
cepa L. test in relation to the average root weight. * = significant
difference in relation to the control group
Fig. 3 Evaluation of the toxicity of three plant extracts using the A.
cepa L. test in relation to the average weight gain of the bulbs.
* = significant difference in relation to the control group
through hydrodistillation. Despite such discrepancy, the
extraction method used in the present work allows the
dragging of a certain amount of components present in the
essential oil, thus allowing to infer plausible comparisons
with the toxicity results presented by the aforementioned
authors. Regarding the toxicity of S. carinatum, the results
presented here agree with those demonstrated by Machado
et al. (2011). These authors evaluated the in vitro cytotoxicity of S. carinatum latex in cultures of bovine fetal
trachea cells and revealed that such component is potentially toxic in high concentrations, however, inducing less
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Fig. 4 Evaluation of the toxicity of three plant extracts using the A.
cepa L. test in relation to the average root size. * = significant
difference in relation to the control group
severe or imperceptible toxic damage, in medium or low
concentrations.
After carrying out the ovicidal test against Ancylostomidae, it is possible to verify that all tested extracts showed
a moderate potential for such purpose, since in all the
evaluated concentrations, they induced a significant inactivation or destruction of eggs, but not enough to induce
100% egg mortality (Table 2).
In the present study, although all the extracts evaluated
did not induce 100% mortality or inactivation of the eggs,
the moderate ovicidal activity observed highlights the
potential of using them for environmental decontamination,
considering that, according to Rey (2001) nematode eggshells are one of the most resistant biological structures,
most substances being impermeable, except for gases and
lipid solvents.
The eggshell of a nematode can contain from one to five
layers, and although there are considerable variations in
their structure, it is known that the inner lipid layer is
responsible for the extreme impermeability of eggs of
certain species, particularly when passing water, which
would justify the partial effectiveness with respect to ovicidal activity, since the extracts that were evaluated are of
hydroalcoholic nature.
It is noteworthy that the transmission of hookworm
occurs predominantly through the penetration of larvae
through the skin of the host, and, for these larvae to carry
out the transmission, they need to hatch, leading, consequently, to the loss of protection that the egg membrane
provides. Thus, it is possible to theorize that greater efficacy of the evaluated extracts would be observed when
determining larvicidal activity.
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Table 2 Ovicidal test against Ancylostomidae using extract of T. minuta, S. carinatum and E. milli at 100 lL/mL, 50 lL/mL and 12.5 lL/mL
concentrations
Tagetes minuta L.
Concentration
Mortality
100.0 lL/mL
28/40 (70%)a
24/40 (60%)
b
12.5 lL/mL
Negative control
22/40 (55%)
0/30
b
Positive control
30/30
50.0 lL/mL
Synadenium carinatum Boiss
Euphorbia milli var splendens
Concentration
Mortality
Concentration
Mortality
100.0 lL/mL
29/40 (72.5%)a
50.0 lL/mL
100.0 lL/mL
24/40 (60%)b
a
50.0 lL/mL
23/40 (57%)b
b
28/40 (70%)
12.5 lL/mL
Negative control
20/40 (50%)
0/30
12.5 lL/mL
Negative control
21/40 (52.5%)b
0/30
Positive control
30/30
Positive control
30/30
a,b
Equal letters imply similarity, in statistical terms
The susceptibility of Ancylostomidae larvae to plant
extracts has been evaluated and demonstrated by several
authors, including Santos et al. (2013). These authors
evaluated the ovicidal and larvicidal activity of 10 plant
extracts of known toxic species, and of these, four namely,
Allamanda cathartica L. (Apocynaceae), Nerium oleander
L. (Apocynaceae), Mirabilis jalapa L. (Nyctaginaceae) and
Brugmansia suaveolens Willd. (Solanaceae), showed
promising activity, with emphasis on the last two, as they
showed larvicidal activity at a concentration of 12.5 mg/
mL.
It should be noted that Santos et al. (2013) were not
successful in evaluating the ovicidal activity of the extracts
that they evaluated, highlighting the evidence of the
resistance of Ancylostomidae eggs and allowing highlighting the potential of the ovicidal activity of all the
extracts tested in the present work, particularly with regard
E. milli var splendens, due to the fact that the hydroalcoholic extract of this plant species, in addition to inducing
the inactivation of eggs in all concentrations evaluated,
showed no significant toxicity in the lowest concentration,
namely 12.5 lL/mL, in both toxicity tests that were outlined, thus implying a risk/benefit probably more favorable.
Another factor that may have influenced the moderate,
but not high, ovicidal activity of the evaluated extracts,
may be a consequence of the fact that in the present work
hydroalcoholic extracts were evaluated, whose extraction
methods may have induced a loss by evaporation of the
components present in essential oils. According to Marotta
et al. (2016) the lipophilia of the compounds presented in
essencial oils, particularly the terpenic components, play an
important role in the cellular damage and structural changes of the cellular membrane, modificating its permeability
and causing the leakage of ions and other cell contents.
Regarding S. carinatum, it is known that the latex of this
plant species has immunomodulatory activity in infections
in murine models against protozoa, such as Toxoplasma
gondii, Neospora caninum and Leishmania amazonensis
(Souza et al. 2016; Cardoso et al. 2012; Afonso-Cardoso
123
et al. 2007), but little is known about its toxicity to helminth eggs, allowing to infer, based on the bibliographic
review outlined in the present research, that such promising
results are unprecedented.
On the other hand, T. minuta ovicidal activity against
nematodes has already been demonstrated by Massuh et al.
(2017), against the phytenomatoid Meloidogyne javanica,
testing the essential oil, which showed 90% suppression of
eggs of this species. Other researchers (Giarratana et al.
2017) demonstrated larvicidal activity of T. minuta essential oil against Anisakis spp. larvae, showing promising
antihelminthic activity against this species of nematode,
however, as well as for the other species evaluated, as far
as extended the bibliographic review outlined in the present
research, there are few articles that mention their in vitro
ovicidal activity against helminths of medical-veterinary
importance, particularly with regard to species of the
Ancylostomidae family.
Conclusion
Based on the results obtained, it can be concluded that the
use of Euphorbia milii var splendens, Tagetes minuta L.
and Synadenium carinatum hydroalcoholic extracts at
100 lL/mL, 50 lL/mL and 12.5 lL/mL concentrations is
of high potential for the control of environmental contamination, and consequently, for the primary prevention
of hookworm, particularly with respect to the extract of E.
mili at a concentration of 12.5 lL/mL, since it presented a
significantly lower toxicity than that of the other plant
species evaluated. It should be noted, however, that new
experiments must be defined, in order to test extracts
obtained with solvents of different polarities, or to test
isolated active principles, in order to maximize the ovicidal
activity and allow a greater potential for applicability in the
control of hookworm.
J Parasit Dis (Jan-Mar 2021) 45(1):252–257
Acknowledgements We thank UniFUNVIC for the opportunity to
outline the experiments in the laboratories involved.
Compliance with ethical standards
Conflict of interest All authors declare that: 1. The manuscript was
not submitted to other journal; 2. The work is original; 3. There isn’t
any plagiarism; 4. Results are presented without fabrication, falsification or inappropriate data manipulation. 5. All the results obtained
are disposed in this publication, that is, it was not splitted out.
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