IJMBR 5 (2017) 1-7
Ethanolic extracts of different fruit trees and their
Letícia Aparecida Duart Bastos
Multidisciplinary Center of Chemical Biological and Agricultural Research (CPQBA), Campinas State University
São Paulo State University (UNESP), Araraquara, SP, Brazil.
Received 05 December, 2016
Received in revised form 02
Accepted 05 January, 2017
Strongyloides venezuelensis and Strongyloides ratti
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Strongyloidiasis is an intestinal neglected disease caused
by the nematode Strongyloides stercoralis with over 100
million estimate cases worldwide (Bisoffi et al., 2013).
The risk of infection is greater in regions with hot and
*Corresponding author. E-mail: email@example.com. Tel: (19)
humid climate, in people who work with soil and/or
et al., 2009).
characteristic of S. stercoralis lies on the ability to
replicate inside the host and auto-infect it. Autoinfection
may lead to persistent chronichyper infections, with a
wide variation of chronic hyper manifestations, which
et al., 2006) with an 87% mortality rate (Olsen et al.,
Strongyloides venezuelensis and Strongyloides ratti,
parasites, are important models both in an
immunologic and biologic perspective, for the develop-
ment of new drugs and diagnostic tests (Nolan et al.,
1993; Olson and Schiller, 1978; Sato and Toma, 1990).
Nowadays, strongyloidiasis treatment is performed with
either albendazole (400 mg/kg) or ivermectin (200 µg/kg)
(Olsen et al., 2009; WHO, 2012). Thiabendazole,
mebendazole and cambendazole, have been used for
strongyloidiasis treatment, however their use has been
dropped due to their toxicity and adverse effects, frequent
treatment fail and drug resistance (Bisoffi et al., 2013;
Legarda-Ceballos et al., 2016; Olsen et al., 2009).
Due to high prevalence and low therapeutic efficiency
of synthetic drugs currently available for strongyloidiasis
treatment, there is a need for new therapeutic alter-
natives. Medicinal plants and their extracts have been
used in the treatment of several diseases, including
parasite infections, making them a viable alternative in
the search for new drugs against S. stercoralis (Anthony
et al., 2005).
In this report, the anthelminthic in vitro effect of
ethanolic extracts, obtained from several species of
Brazilian fruit trees, against S. venezuelensis parasitic
females is described; in an attempt to search for new
MATERIALS AND METHODS
Plant leaves were collected in the Multidisciplinary Center
(CPQBA) at Campinas State University (UNICAMP), in
Paulínia, SP. 24 plants were used (Table 1). The criteria
for selection of plant was to use only plants that bear fruit
and were easy to cultivate.
The ethanolic extracts were obtained by mixing dried
10 min/16.000 rpm in a mechanical disperser (Ultra
Turrax T50, IKA Works Inc., Wilmington, NC, USA),
followed by filtration. The residue was re-extracted with
100 mL of ethanol. The extracts were pooled and
evaporated under vacuum until dry, resulting in the final
dried ethanolic extracts.
The extracts with better results were then fractioned.
Briefly, 1.33 g of dry extract was dissolved in 50 mL of
distilled water with help of a sonicator, transferred to a
Int. J. Mod. Biol. Res. 2
separation funnel and then, 50 mL of acetone were
added. The process was repeated and instead of
acetone, 50 mL of dichloromethane was added. The
organic and aqueous fractions were separated and dried
in a rotary evaporator.
For the control group three synthetic compounds were
used, albendazole (generic drug EMS, 400 mg),
cambendazole (Cambem®, UCI-farma, 180 mg) and
ivermectine (Ivermec®, UCI-farma, 6 mg).
In the research, a S. venezuelensis strain isolated from
tained for several years in our lab at UNICAMP was
used, through successive infections in Rattus norvegicus,
Wistar lineage. The experiments were approved by the
Ethics Commission for Animal Use (CEUA/UNICAMP,
protocol 2174-1), as they were in accordance with the
ethical principles of animal experimentation adopted by
Parasite female recovery
Fifteen days after infection, the rats were euthanized and
longitudinally. The intestine was washed with sterile
saline solution (0.15 mol/L) and placed in a Petri dish
containing RPMI 1640 (Nutricell
) medium (supple-
penicillin, 0.3 g/L of L-Glutamine, 2.0 g/L of D-Glucose,
2.0 g/L of NaHCO
and 5,958 g/L of Hepes), and were
collect and washed three times in RPMI medium to avoid
later contamination during the in vitro assays. Only
parasitic females (pathenogenetic) were used since they
are the main parasitic form living in the vertebrate host
and for their ability to reproduce inside the host which
may lead to a disseminated infection. Males only exist as
a free-living form in the soil.
In vitro assay
Both the extracts and synthetic drugs were diluted in 2%
Bastos et al. 3
three concentrations, 0.05, 0.1 and 0.2 mg/mL. The in
tration was tested three times (n = 6). A control group of
RPMI medium and 2% PBS. The plates were incubated
at 37°C in a 5% CO
atmosphere and observed at 2, 4, 6,
). Motility (Absence, low, moderate and high),
oviposition and mortality were observed.
Inhibitory concentration was determined using Origin 7
program. Significant differences between groups were
calculated using one-way Analysis of Variance (ANOVA)
(p<0.0001). The correlation between time, motility,
oviposition, and mortality with the extract concentration
was accessed using Duncan’s
Multiple Range Test.
The ethanolic extracts yield, from an initial 10 g mass,
Worm motility was compared between the average of
the averages of the extract that show significant effect
(p<0.0001) against S. venezuelensis is highlighted
S. mombin, Psidium cattleianum, Inga cylindrica,
Manilkara zapota, Eugenia pyriformis, Labramia bojeri,
Myrcianthes pungens, Byrsonima crassifolia, Eugenia
brasiliensis, Muntingia calabura, Carya illinoensis,
Hexachlamys edulis, Eugenia uniflora and Pourouma
Int. J. Mod. Biol. Res. 4
Duncan’s Multiple Range Test –
worms mean motility at different observation times: 1-6 h (T1), 12 h (T2), 24 h (T3), 48 h (T4),
e 72 h (T5). Calculated according with the scale: 0, Absence; 1, low; 2, moderate; 3, high.
0.83 (l, k)
2.11 (f, h, e, g)
1.00 (i, g, h)
0.61 (k, j, i, h)
2.77 (a, b)
1.83 (h, g)
1.55 (h, i)
0.55 (j, k)
0.44 (k, j, l)
1.00 (j, k)
0.72 (i, j)
0.16 (m, l)
2.22 (e, d, f)
1.22 (j, k)
0.66 (i, j, k)
2.44 (b, d, c)
0.77 (i, j)
0.55 (k, j, i, l)
1.88 (h, g, f)
1.72 (h, i)
0.61 (i, j, k)
2.72 (b, a, c)
2.22 (f, d, e, g)
0.83 (i, j, h)
0.33 (k, m, l)
2.00 (e, g, f)
0.27 (k, m, l)
1.38 (j, i)
0.50 (k, j, l)
2.27 (f, d, e)
1.27 (f, g)
2.27 (e, d)
2.00 (f, h, g)
1.22 (f, g)
0.77 (g, j, i, h)
1.16 (f, g, h)
2.33 (e, d)
2.61 (b, d, a, c)
1.94 (c, b, d)
1.00 (g, f, h)
2.38 (d, c)
2.38 (f, d, e, c)
0.94 (g, i, h)
2.00 (c, b, d)
1.50 (c, e, b, d)
2.77 (b, a)
2.50 (d, e, c)
1.16 (g, e, f)
2.94 (b, a)
2.22 (c, b)
1.77 (c, b)
1.77 (e, d)
1.38 (c, e, d)
2.77 (b, a, c)
1.83 (c, d)
1.55 (c, e, b, d)
1.61 (c, b, d)
2.55 (b, d, c)
1.44 (f, e)
1.33 (e, f, d)
EE, Ethanolic extract; FA, aquous fraction; FO, organic fraction; DC, drug control; Cont, RPMI control.
S. mombin ethanolic extract and aqueous fraction
for all tested concentrations. Overall most of the extracts
showed a satisfactory anthelmintic effect for at least one
of the tested concentrations (Table 3): P. cattleianum, M.
100% mortality at 0.2 e 0.1 mg/mL. Litchi chinensis,
ethanolic extracts did not show activity.
With the purpose of isolating and identifying the
chemical compounds that act as antheminthics, S.
extraction, resulting in an organic and an aqueous
fraction. The results showed a higher efficiency for the
organic fraction killing 100% of all females at all tested
concentrations, whilst the aqueous fraction only showed
activity in the two highest concentrations (0.2 mg/mL e
Cambendazole, albendazole and ivermectin did not kill
any female worm during the observation period for the
tested concentrations. No correlation was found between
the plant family and their anthelminthic activity.
Bastos et al. 5
mg/mL); C3 (0.05 mg/mL).
EE, Ethanolic extract; FA, aquous fraction; FO, organic fraction; DC, drug control.
Strongyloidiasis is a silent, underdiagnosed and the most
Currently, there is no satisfactory drug in the treatment of
this parasite, since its efficiency varies between different
patients (Bisoffi et al., 2013). Both efficient and inefficient
treatment are often reported in the same regions with the
same treatment scheme (Panic et al., 2014), there is,
therefore a need to search for new drugs. The use of
medicinal plants in the search of new drugs is increasing,
and several plants have shown their anthelminthic activity
(Muthee et al., 2011). Plants active compounds
identification has been increasing, contributing for a
higher variability and availability of drugs and bringing
highly accepted therapeutic alternatives (de Oliveira et
al., 2014). The majority of published studies test the in
2008; Kotze et al., 2004), to our knowledge, this is the
first in vitro study investigating the anti-Strongyloides
activity against adult parasitic females. Our methodology
was derived from the one published by de Oliveira et al.
(2012), where Schistosoma mansoni was used as an
experimental model, and provided an efficient way to
perform in vitro tests with S. venezuelensis.
Promising results of some plant species anti-parasite
effect against parasites such as S. mansoni (de Oliveira
et al., 2014; de Oliveira et al., 2012) and Giardia
duodenalis have been described (Machado et al., 2011;
Muthee et al., 2011). Their use as also been researched
against other organisms, such as fungus (Wianowska et
al., 2016), virus (Gonçalves et al., 2005) and tumors
pharmacologic action of most plants used in this paper.
Among the tested extracts, S. mombin showed the most
promisingresults. Several other authors have been
exploring the activity of S. mombin ranging from their use
against the diarrhea rotavirus (Gonçalves et al., 2005),
against larvae and adult mosquitos (Ajaegbu et al., 2016;
Eze et al., 2014), Candida albicans (Okwuosa et al.,
2012), Leishmania chagasi (although it has shown low
activity against L. amazonensis amastigote) (Accioly et
al., 2012; Estevez et al., 2007), anti-bacterial activity (da
Silva et al., 2012), and activity against Eudrilus eugeniae
(annelida, Oligochaeta) (Gbolade and Adeyemi, 2008). S.
mombin anthelminthic activity has also been reported
against sheep nematode (Ademola et al., 2005) and
against small ruminants gastro-intestinal parasites in
Benin (Attindéhou et al., 2012). It is also reported that S.
2006), showing a wide variety of potential uses for this
plant. Several other extracts showed promising results, I.
cylindrica showed the best results after S. mombin and
may represent a potential drug candidate.
Ivermectin and albendazole present irregular cure rates
(55 - 100% and 38 - 87%, respectively) as well as several
side effects, showing a need for new drugs to be
developed. However very few papers have been
published, and even fewer with promising results
(Boonmars et al., 2005; de Oliveira et al., 2014; Keiser et
al., 2008; Kotze et al., 2004; Legarda-Ceballos et al.,
2016; Olounlade et al., 2012). In this paper, we show that
the majority of the extracts had a higher in vitro efficiency
than the drugs currently used for the treatment of
strongyloidiasis, with emphasis on S. mombin and I.
cylindrica that showed the best results. S. mombin
organic fraction showed great potential and should be
further studied to isolate and identify their active
compounds, which are responsible for the anthelminthic
activity so that, hopefully, a new drug can be developed
to fight against strongyloidiasis.
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