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Efficacy of aqueous extracts of Genipa americana

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and eclosion of gastrointestinal nematodes of
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Efficacy of aqueous extracts of Genipa americana

L. (Rubiaceae) in inhibiting larval development and
eclosion of gastrointestinal nematodes of sheep
a

Flvia Aparecida Nogueira , Patrcia Silva Nery , Franciellen Morais-Costa , Neide Judith de
a

Faria Oliveira , Ernane Ronie Martins & Eduardo Robson Duarte

Instituto de Cincias Agrrias, Universidade Federal de Minas Gerais, Montes Claros, Brazil
Published online: 12 Feb 2014.

To cite this article: Flvia Aparecida Nogueira, Patrcia Silva Nery, Franciellen Morais-Costa, Neide Judith de Faria Oliveira,
Ernane Ronie Martins & Eduardo Robson Duarte (2014) Efficacy of aqueous extracts of Genipa americana L. (Rubiaceae) in
inhibiting larval development and eclosion of gastrointestinal nematodes of sheep, Journal of Applied Animal Research, 42:3,
356-360, DOI: 10.1080/09712119.2013.845103
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Journal of Applied Animal Research, 2014

Vol. 42, No. 3, 356360, http://dx.doi.org/10.1080/09712119.2013.845103

SHORT COMMUNICATION
Efficacy of aqueous extracts of Genipa americana L. (Rubiaceae) in inhibiting larval
development and eclosion of gastrointestinal nematodes of sheep
Flvia Aparecida Nogueira, Patrcia Silva Nery, Franciellen Morais-Costa, Neide Judith de Faria Oliveira,
Ernane Ronie Martins and Eduardo Robson Duarte*
Instituto de Cincias Agrrias, Universidade Federal de Minas Gerais, Montes Claros, Brazil

Downloaded by [Franciellen Morais-Costa] at 07:58 04 November 2014

(Received 15 April 2013; accepted 11 September 2013)

The anthelminthic efficacy in vitro of the aqueous extract of Genipa americana L. leaves was evaluated at final
concentrations of 20, 30, 50, 75 and 100 mg (dry matter)/mL through egg hatching inhibition (EHI) tests and
quantitative cultures. The observed lethal concentration LC90 values for hatching and L3 development inhibition were
79.8 and 28.7 mg/mL, respectively. The extract was more effective in larval development inhibition (LDI) than in
hatching inhibition. Phytochemical analysis revealed tannins and flavonoids in the extract. The utilisation of
G. americana as an anthelminthic may represent a viable alternative to synthetic products, although further studies
are needed to verify its in vivo efficacy and to assess its toxicity in sheep.
Keywords: anthelminthic; quantitative coproculture; egg hatching inhibition; phytotherapy; small ruminants

1. Introduction
Demand for ovine meat has grown, mainly in large urban
centres, and sheep farmers have endeavoured to optimise
production. For success, it is important to ensure the
health of animals. Haemonchosis is a major disease in
sheep, affecting both sexes at all age levels, reducing
weight gain and reproductive capacity, as well as milk,
wool and hide production (Pires et al. 2000; Bizimenyera
et al. 2006).
Gastrointestinal nematode (GIN) control has relied
mainly on frequent use of commercial anthelminthic
drugs. However, the emergence of drug resistant GIN
populations (Jackson & Coop 2000; Melo & Bevilaqua
2002) has stimulated research into alternative approaches,
including phytotherapy (Githiori et al. 2006).
The efficacy of anthelminthic compounds must be
preserved through judicious use. Research into new
anthelminthic molecules that may be used along with
current drugs is laborious and costly, and the time span
from trials to commercial availability is lengthy (Hennessy 1997). Research into alternatives for the control of
GIN is essential.
Plant secondary compounds can be used as alternatives to control GINs in ruminant livestock (Kahn &
Diaz-Hernandez 2000). However, scientific validation of
the anthelminthic effects and investigation into possible
side effects are necessary prior to the adoption of plant
extract therapies (Githiori et al. 2006).
The genipap (Genipa americana L.) is a rubiaceous
tree that has been used in folk medicine, foods and
*Corresponding author. Email: duartevet@hotmail.com
2014 Taylor & Francis

animal feed, leather tanning, forestry and logging industries. The species, native to South America, has ecological importance for feeding animals, and is suitable for
planting in degraded areas and wetlands (Epistein 2001).
The use of medicinal plants for nematode control can
reduce the presence of chemical residues in animal
products and the development of nematodes with
anthelminthic resistance (Hammond et al. 1997). The
utilisation of these extracts for the reduction of anthelminthic-resistant populations of ovine nematodes may
constitute a promising strategy in herds with high
frequency of anthelminthic multiresistance (Nery et al.
2010). Although G. americana is not included as an
anthelminthic in folk use, it is a plant rich in tannins
(Hernes & Hedges 2004) and therefore, the goal of the
present study was to evaluate in vitro the anthelminthic
efficacy of G. americana on the inhibition of larval
development and eclosion for GIN-infecting sheep.

2. Materials and methods

Leaves, flowers and fruits of G. americana were
collected in a rural region of the Cerrado, Montes Claros
City, northern Minas Gerais, Brazil (165518S 4352
11W), in January 2008, during the morning. Identification was based on Almeida et al. (1998) and Lorenzi
(1998). Specimens were deposited at the herbarium of
the Montes Claros State University in Brazil, number
HMC 1490.

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Journal of Applied Animal Research

Healthy leaves were selected and dried to constant
weight in a forced air circulating drier at 40 5C. Dried
leaves were ground and stored at 5 3C until use.
Extracts were obtained using the method of Nery et al.
(2010) with modifications. Ground dried leaves were
incubated in a distilled water bath at 60C for 60 min
and hot filtered through a gauze funnel. The aqueous
extract was again dried in a forced air oven at 40C.
Tests to determine the main secondary metabolites in
the extracts were conducted using the colorimetric
method described by Matos (1997) and Maciel et al.
(2006). Aliquots of the aqueous extract were diluted in
distilled water. Tannins were identified with lead acetate,
copper acetate and glacial acetic acid reactions. Phenols
were identified by the ferric chloride test and flavonoids
by the Shinoda method, ferric chloride and sodium
hydroxide tests. Steroids and terpenoids were confirmed
by the Lieberman-Burchard reaction; alkaloids using
Dragendorff, Mayer and Bourchard reagents; and saponins using the foam test (Matos 1997; Maciel
et al. 2006).
2.1. In vitro egg hatching inhibition test
The dry extract was dissolved 1:3 in distilled water at
40C for 30 min. The mixture was filtered through gauze
and cotton and stored at 4C until use. Five aliquots of
this solution were performed to dry matter determination
at 105C. The stored leaf aqueous extract solution was
standardised at 200 mg of dry matter/mL and diluted in
sterile distilled water to final concentrations of 40, 60,
100 and 150 mg/mL. Extract solutions were used
immediately in the egg hatching inhibition (EHI) tests
with four replicates (Coles et al. 1992).
Sedimentation in water, filtration and flotation in
saturated saline were used to obtain nematode eggs from
faeces of naturally infected Santa Ins ewes with an
average faecal egg count of >300/g, determined by the
modified McMaster technique (Gordon & Whitlock 1939).
Experimental mixtures comprised 300 L faecal
suspension with an average of 600 fresh eggs combined
with either 300 L of the extract concentrations, 300 L
sterile distilled water for the negative control or 300 L
of ivermectin suspension (16 g/mL) as positive control.
All experimental mixtures were homogenised and incubated in a BOD incubator at 28C for 48 h. Subsequently, 15 L Lugols solution was added to each tube,
which were stored at 4C for counting of unembryonated
and embryonated eggs and first stage larvae (L1; Coles
et al. 1992).
The number of L1 relative to the total number of
eggs plus L1 was determined for each repetition and
subjected to variance analysis. The means were compared using the ScottKnott test at 5% significance.

357

Probit regression was employed to determine the concentrations sufficient to inhibit 50% (lethal concentration, LC50) and 90% (lethal concentration, LC90) of egg
hatching using the statistical package, Saeg 9.1 (2007).
The formula of Coles et al. (1992) was used to determine
the EHI effectiveness:


1  mean of L1
% effectiveness 100 
mean eggs L1
2.2. In vitro larval development inhibition
Faeces of five naturally infected Santa Ins crossbred
lambs, age six to eight months and producing >500 eggs/g
of faeces were used. The leaf extract solution was
prepared as described, standardised at 200 mg/mL, and
diluted in sterile distilled water to 40, 60, 100 and
150 mg/mL. The extracts were used immediately in a
larval development inhibition (LDI) test in quantitative
culture (Borges 2003; Nery et al. 2010).
Unpreserved faeces collected directly from the rectal
ampulla of each animal were pooled and divided into 2 g
samples distributed among clean disposable cups. Two
mL of each extract, 2 mL of ivermectin solution (Ranger
LA, Valle, Montes Claros, MG, Brazil) equivalent to 16
g/mL (positive control), or 2 mL sterile distilled water
(negative control) were added to the faeces. Each
concentration and the controls were evaluated in four
replications
On day 7 of the culture, the nematode larvae were
collected in a test tube and held at 4C until counting.
To identify the genus, slides were prepared with Lugols
iodine, and identified using the key by Keith (1953). The
number of L3 was divided by two to give the number of
L3 per gram of faeces (LPGF). The following formula,
adapted from Borges (2003), was used to determine the
percent reduction in larva numbers per gramme of
faeces:


1  LPGF treated group
% effectiveness 100 
LPGF negative control
The data were transformed, log (x + 1), and submitted
to variance analysis. The means were compared by the
ScottKnott test and 5% probability calculated. The LC50
and LC90 were determined by probit analysis using the
statistical package Saeg 9.1 (2007).
Experimental procedures with sheep were carried out
in accordance with the Experience Ethical Committee of
Minas Gerais Federal University CETEA UFMG and
approved by this committee under protocol number
042-2008.
3. Results and discussion
The phytochemical analysis of aqueous extract confirmed the presence of flavonoids and tannins. The

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358

F.A. Nogueira et al.

addition of glacial acetic acid to lead acetate indicated

the predominance of soluble tannins, since this reaction
is specific to hydrolysable tannins.
The tannins and flavonoids are secondary metabolites
with therapeutic potential. A recent study showed that a
flavonoid-containing aqueous extract of immature mango
is active against Haemonchus contortus (Nery et al.
2012). Tannins can complex with proteins, reducing
available amino acids and resulting in larval death by
starvation, or can bind to the glycoprotein-rich cuticle of
the larvae, causing death (Athanasiadou et al. 2001).
Reduction in the number of nematode eggs in faeces of
sheep fed tanniferous plants has been shown by Marley
et al. (2003), Lange et al. (2006) and Minho et al. (2008).
Other hypotheses may explain the in vivo anthelmintic
action of tannins. These compounds can bind dietary
proteins and protecting them from ruminal degradation,
increasing protein flux and amino acid absorption in the
small intestine, favouring the immune response against the
worms (Hoste 2006). Tannin intakes can have negative or
positive effects on animal productivity (Frutos et al 2004),
depending on which and how much is consumed the
compounds structure and molecular weight, and on the
physiology of the consuming species (Hagerman & Butler
1991). Thus, studies of the tannin characterisation and
toxic analysis of acute or chronic ingestion be conducted
carefully for treatment of helminthiasis in ruminants.
In the EHI test, the aqueous extract at 100 mg/mL
completely inhibited hatching. The relative mean number
of embryonated eggs was significantly greater than
unembryonated eggs at 75 and 100 mg/mL (P < 0.05).
This suggests greater efficacy in inhibiting hatching than
in interfering with embryo development (Table 1). Probit
analysis showed the LC50 and LC90 of aqueous extract of
G. americana leaves to be 34.3 and 79.8 mg/mL,
respectively. Culture and identification showed 99.8%
of the L3 larvae to be Haemonchus spp., and 0.2% were
Trichostrongylus spp.
In the LDI test, concentrations 30 mg/mL showed
anthelminthic efficacy above 94% (Table 2). Based on

Table 2. Mean nematode L3 count/g of faeces (LPGF) in

coprocultures treated with aqueous extract of leaves of
G. americana and efficacy in larval development inhibition.
Concentrations
(mg/mL)
100
75
50
30
20
Water
Ivermectin

Mean
viable (LPGF)
0
1
1
4
19
78
0

0.0c
0.5c
0.5c
0.53b
28.3b
34.6a
00c

Efficacy*
(%)
100.0
99.6
99.6
94.5
74.9

100.0

Note: Different lower case letters indicate significant differences. (P <

0.05) in ScottKnott test. Coefficient of variation = 23.5%.
*Efficacy (%) = 100 (1 LPGF of treated group/LPGF of negative
control).

probit analysis, the LC50 and LC90 were 14.6 and 28.7
mg/mL, respectively. The L3 larvae were 84.7% Haemonchus, 13.3% Trichostrongylus and 2% Strongyloides. This suggests that the extracts were effective
against several nematodes considered to be the most
prevalent and pathogenic in sheep (Wood et al. 1995),
showing a wide spectrum of action.
Both the EHI and LDI showed the G. americana
extract to have anthelminthic activity, suggesting action
on two life stages of nematodes. The presence of tannins
and flavonoids was detected in the leaf extract. Simes
et al. (1999) stated that possible interaction and synergism between the active vegetal metabolites should not
be ignored.
The 90% lethal concentrations for inhibiting larval
development and hatchability were 28.7 and 79.8 mg/
mL, respectively. These data suggest higher efficacy of
the aqueous extract for inhibition of larval development
than for inhibition of eclosion. However, using the
hydro-alcoholic extract of leaves of genipap, KrychakFurtado (2006) found 100% efficacy for EHI at 50 mg/
mL, suggesting that the metabolites extracted with
alcohol could also show action against nematode eggs.

Table 1. Efficacy of aqueous extracts of G. americana leaves on EHI of nematodes from sheep.
Concentrations (mg/mL)
100
75
50
30
20
Water
Ivermectin

Unlarvaed egg (mean)

Larvaed egg (mean)

L1 (mean)

Total eggs + L1

Efficacy* (%)

162.5
90.0
72.5
50.0
112.5
0.0
450.0

662.5
447.5
85.0
52.5
110.0
0.0
0.0

0.0
100.0
308.0
357.5
292.5
360.0
157.5

825.0
637.5
465.5
460.0
515.0
360.0
607.5

100.0a
84.3b
40.1c
33.2c
43.2c
0.0d
74.1

Note: Different lower case letters indicate significant differences. (P < 0.05) in Scott Knott test. Coefficient of variation = 30.4%.
*Efficacy (%) = 100 (1mean of L1/ mean eggs + L1).

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Journal of Applied Animal Research

Other in vitro studies have shown promising results
for vegetal extracts. In tests of anthelminthic activity of
Melia azedarach leaves, the aqueous and hydro-alcoholic extracts at 12.5 mg/mL inhibited 99.4% and 100%
of egg hatching, respectively (Kamaraj et al. 2010).
Ethanolic and dichloromethane extracts of Phytolacca
icosandra produced in vitro activity against the H.
contortus greater than 90% in EHI tests when used at
concentrations of 0.90 mg/mL or higher (HernndezVillegas et al. 2011).
The present study used the modified coproculture test
in which the extract was added to the faeces, the natural
environment for incubation and development of larvae,
increasing the accuracy and precision of the method.
Results from applying the extract directly to the faeces
cannot be accurately extrapolated to the treatment of
animals via ingestion of the plants. However, as reported
in previous studies, the modified coproculture test may
better approximate treatment by ingestion than does the
in vitro EHI test, which is conducted in distilled water
(Nery et al. 2012; Nogueira et al. 2012).
Using the coproculture quantitative test, Nery et al.
(2010) reported that the aqueous extract of Anacardium
humile leaves at 150 mg/mL was 97.3% effective, and
the ethanol extract at 80 mg/mL was 99.6% effective, in
LDI. In similar research, the LC90 for LDI of aqueous
extract of Caryocar Brasiliense was 53.19 mg/ml.
(Nogueira et al. 2012) and the fresh juice of immature
mangos showed greater than 95% efficacy in LDI at 44.4
mg/mL (Nery et al. 2012), Using conventional in vitro
test, aqueous extracts of Leonotis ocymifolia, Leucas
martinicensis, Albizia schimperiana and Senna occidentalis induced more than 96% efficacy at 50 mg/ml
(Egualea et al. 2011). Comparing these results, in this
study the LC90 for LDI was only 28.7 mg/mL, suggesting the extract of G. americana as more effective for
inhibiting the development of larval nematodes of sheep
than these other plants.
In addition, the production of an aqueous extract of
genipap leaves is accessible by sheep farmers in tropical
regions, which could lower costs and reduce risks posed
by chemicals to animals, humans and the environment.
These results, together with future studies evaluating the
acute and chronic toxicity in sheep and in vivo efficacy,
may contribute to the use of this plant species as an
alternative to synthetic anthelminthics.

4. Conclusion
The aqueous extract of G. americana leaves is effective
for both inhibition of larval development and hatchability of GIN in sheep. The LC90 for inhibition of larval
development is 28.7 mg/mL and the extract is more
effective against larvae than eggs.

359

Acknowledgements
We thank the Banco do Nordeste of the Brazil for providing
financial support. The authors are also grateful to Coordenaco
de Aperfeioamento de Pessoal de Nvel Superior CAPES,
Braslia, DF, Brazil, Pro-Reitoria de Pesquia da Universidade
Federal de Minas Gerais and the Fundao de Amparo
Pesquisa de Minas Gerais FAPEMIG.

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