In Vitro Activity of Lantana Camara, Alpinia Zerumbet, Mentha Villosa And

Home , Tagetes

Veterinary Parasitology 190 (2012) 504–509

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

Contents lists available at SciVerse ScienceDirect provided by Elsevier - Publisher Connector

Veterinary Parasitology

jou rnal homepage: www.elsevier.com/locate/vetpar

In vitro activity of Lantana camara, Alpinia zerumbet, Mentha villosa and

Tagetes minuta decoctions on Haemonchus contortus eggs and larvae

a a,∗ a

Iara T.F. Macedo , Claudia M.L. Bevilaqua , Lorena M.B. de Oliveira ,

a a b

Ana L.F. Camurc¸ a-Vasconcelos , Selene M. Morais , Lyeghyna K.A. Machado ,

Wesley L.C. Ribeiro

Programa de Pós-Graduac¸ ão em Ciências Veterinárias, Universidade Estadual do Ceará, Brazil

Rede Nordeste de Biotecnologia, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:

The resistance of gastrointestinal nematodes to anthelmintics has increased the need to

Received 10 November 2011

evaluate natural products that can replace or assist current strategies to control gastroin-

Received in revised form 22 June 2012

testinal nematodes. The objective of this study was to evaluate the effect of decoctions

Accepted 2 July 2012

of Lantana camara (DLc), Alpinia zerumbet (DAz), Mentha villosa (DMv) and Tagetes minuta

(DTm) on Haemonchus contortus by two in vitro tests. The effects of increasing concentra-

Keywords:

tions of lyophilized decoctions (0.31 to 10 mg/ml) were assessed using the egg hatch test

Phytotherapy

Anthelmintic (EHT). The decoctions were then tested in the larval artiﬁcial exsheathment assay. H. contor-

tus third stage larvae (L3) were exposed to 0.31 mg/ml A. zerumbet and M. villosa decoctions

Gastrointestinal nematodes

Tannins and 0.62 mg/ml T. minuta and L. camara decoctions for 3 h and then exsheathment procedure

at 10 min intervals. An inhibitor of tannins, polyvinyl polypyrrolidone (PVPP), was used to

study if tannins were responsible for the inhibitory effect on hatching and exsheathment

of larvae. A. zerumbet, M. villosa and T. minuta showed a dose-dependent effect in the EHT,

which did not disappear after the addition of PVPP. No effect was observed for L. camara

in the EHT. However, the decoctions inhibited the process of larval exsheathment, which

may be related to tannin action because the addition of PVPP reversed the inhibitory effect.

A. zerumbet, M. villosa and T. minuta decoctions showed inhibitory activity on H. contortus

larvae hatching and exsheathing. The decoctions of these plants could be used to control

gastrointestinal nematodes following conﬁrmation of their anthelmintic activity in vivo.

1. Introduction food consumption, decreased milk production, low fer-

tility and, in cases of massive infections, high mortality

The diseases caused by gastrointestinal nematode para- rates (Nunes et al., 2007). The high prevalence associated

sites occupy a prominent place among the factors limiting with pathogenicity make the hematophagous nematode

sheep and goat production and are responsible for high eco- Haemonchus contortus the main parasite of small rumi-

nomic losses due to delayed growth, weight loss, reduced nants in Brazil (Amarante, 2004). The control of parasitism

is accomplished primarily through the use of commer-

cial semi-synthetic and synthetic anthelmintics. However,

these drugs are associated with drawbacks, the risk of

∗

Corresponding author at: Programa de Pós-graduac¸ ão em Ciências

residues in food and environmental contamination (Waller,

Veterinárias/FAVET/UECE, Av. Dede Brasil, 1700, Campus do Itaperi, CEP

2006). Moreover, misuse and indiscriminate treatment

60714-903, Fortaleza, Ceará, Brazil. Tel.: +55 85 31019853;

with synthetic drugs have allowed the rapid selection of

fax: +55 85 31019840.

E-mail address: [email protected] (C.M.L. Bevilaqua). resistant helminth populations (Melo et al., 2003). Thus, it

I.T.F. Macedo et al. / Veterinary Parasitology 190 (2012) 504–509 505

has become necessary to develop studies aimed at search- 2.2. Obtaining H. contortus eggs and larvae

ing for complementary alternatives to traditional methods

(Ademola and Eloff, 2010). One 6-month old, male sheep, of mongrel breed, was

The use of plants with anthelmintic properties seems kept in a metabolic cage and initially treated with three

to be an effective alternative, both from the standpoint anthelmintics with different active ingredients (Fenben-

® ®

of parasite control and their low environmental impact dazole, Panacur ; Levamisole, Ripercol , and Ivermectin,

(Hammond et al., 1997). The natural products are a mix- Ivomec )on alternate days to eliminate gastrointestinal

ture of components which act in synergy producing the nematodes. Five thousand infective larvae (L3) of H. con-

anthelminthic effect, differing from the commercial drugs tortus benzimidazole-resistant were orally inoculated 21

which have one molecule acting on the parasite when days after treatment. Subsequently fecal samples were col-

not combination formulation. Thus, the resistance is likely lected to conﬁrm the establishment of the experimental

to develop more slowly in the natural product. Plant infection, using a modiﬁed McMaster technique, obtaining

extracts have been used since antiquity as remedies for an egg count per gram of feces (epg) above, 2000.

many diseases. Infusions and aqueous decoctions are the To recover H. contortus eggs, 10 g of feces collected

main methods of preparation used in traditional medicine directly from the rectum of the experimentally infected

(Scudeller et al., 2009). Among the species of plant sheep was processed according to the technique described

that have been documented as having medicinal effects by Hubert and Kerboeuf (1992). L3 were collected from

against helminths are Lantana camara (Verbenaceae), feces according to Ueno and Gonc¸ alves (1998).

Tagetes minuta (Asteraceae), Mentha villosa (Lamiaceae)

(Albuquerque et al., 2007) and Alpinia zerumbet (Zingiber- 2.3. Egg hatch test (EHT)

aceae) (Almeida, 1993). Recent studies have suggested that

plants containing condensed tannins may offer a promis- This test was performed based on the methodology

ing alternative approach to control parasitism (Rochfort described by Coles et al. (1992). To increase the solubility in

et al., 2008). Condensed tannins are secondary plant aqueous medium, the decoctions were diluted in 3% Tween

metabolites that comprise the most widespread class in 80. An egg suspension (250 ␮l) containing approximately

␮

nature. 100 fresh eggs were incubated with 250 l decoctions at

◦

The value and credibility of herbal medicines depend ﬁnal concentrations of 0.31–10 mg/ml for 48 h at 25 C.

on the recognition of the healing properties of some Drops of Lugol were added, and the eggs and ﬁrst stage

plants. Thus, scientiﬁc experimentation is a crucial larvae (L1) were counted under a microscope. This test

and obligatory step to prove the effectiveness of had two controls: a negative control containing the dilu-

plants popularly used as anthelmintics (Githiori et al., ent (Tween 80) and a positive control with 0.025 mg/ml

2006). To assess the anthelmintic properties of plant thiabendazole (diluted with 3% dimethylsulfoxide-DMSO).

extracts, in vitro testing can be used as a preliminary Three repetitions in different days with ﬁve replicates for

step to characterize the possible effects (Costa et al., each decoction concentration and for each control were

2002). performed.

The objective of this study was to evaluate the effects To evaluate the role of tannins on the anthelmintic

of decoctions of L. camara (DLc), A. zerumbet (DAz), M. vil- effect, a speciﬁc inhibitor of tannins, polyvinyl polypyrroli-

losa (DMv) and T. minuta (DTm) on egg hatching and larval done (PVPP; Sigma ), was used. The PVPP binds to tannins

exsheathment of H. contortus. inhibiting from exercising their effect, i.e., allowing the

egg hatch. The highest concentrations of DAz, DTm and

2. Materials and methods DMv were pre-incubated overnight with 50 mg/ml PVPP

(Alonso-Díaz et al., 2008).

2.1. Decoction extraction

2.4. Larval artiﬁcial exsheathment assay (LAEA)

The plants used were collected in the Horto of medic-

inal plants of the Universidade Federal do Ceará in plots This test was based on the methodology described by

exposed to environmental conditions in a tropical coun- Bahuaud et al. (2006). One thousand H. contortus L3 were

try in Fortaleza, State of Ceará, Brazil. All plants were incubated for 3 h in a solution containing decoction diluted

authenticated and voucher specimens were deposited in in phosphate buffer solution (PBS) at concentrations of

␮

the Herbarium Prisco Bezerra of the Universidade Fed- 310 g/ml (DAz and DMv) or 620 ␮g/ml (DLc and DTm)

eral do Ceará, under the numbers: L. camara- 46,017, or the negative control with 1 ml of PBS. Larvae were then

A. zerumbet- 49,659, M. villosa- 52096 and T. minuta- washed and centrifuged three times in PBS and subjected to

49,676. an artiﬁcial exsheathment process by contact with a solu-

For the production of the decoctions, fresh plant tion of sodium hypo chloride (2% w/v) diluted 1:300 in PBS.

material was cut from aerial parts and completely sub- The kinetics of larval exsheathment were monitored micro-

merged in distilled water at 1:10 (g/ml) and boiled for scopically for 1 h to identify the exsheathed larvae at 0, 10,

2 h under heating at reﬂux with reconstitution of the 20, 30, 40, 50 and 60 min. Six replicates were performed for

evaporated volume. Subsequently, the solutions were each decoction and the control to examine the changes in

ﬁltered, frozen and lyophilized. The decoctions stored the proportion of exsheathed larvae in relation to time.

◦

at 4 C until used. The decoctions were soluble in Each decoction concentration was pre-incubated

water. overnight with 50 mg/ml PVPP to evaluate the role of

506 I.T.F. Macedo et al. / Veterinary Parasitology 190 (2012) 504–509

Table 1

Mean efﬁcacy (percentage of reduction inhibition of egg hatching) ± standard error by decoction of Lantana camara (DLc), Alpinia zerumbet (DAz), Tagetes

minuta (DTm) and Mentha villosa (DMv) on Haemonchus contortus egg hatching.

Concentrations (mg/ml) DLc DAz DTm DMv

a a b

0.31 – 12.7 ± 1.2A 12.1 ± 1.77A 24.7 ± 1.74A

a a b c

0.62 18.3 ± 0.84AB 18.5 ± 1.57A 44.6 ± 4.01B 62.7 ± 2.87B

± a b c c

1.25 18.8 0.74AB 74.6 1.96B 85.8 ± 2.4C 90.0 ± 2.18CD

± a b c c

2.5 14.0 1.13A 82.7 ± 1.55C 96.8 ± 0.77D 97.6 ± 0.79C

a b b

5 15.7 ± 1.66AB 97.5 ± 0.84D 100 ± 0.0D –

10 19.7 ± 2.15B – – –

a a a

Pvpp* + decoction – 82.6 ± 1.46C 92.8 ± 2.04CD 84.3 ± 4.5D

a a a a

Tween 80 (3%) (negative control) 13.0 ± 0.77A 14.3 ± 1.05A 13.7 ± 1.84A 11.2 ± 1.46E

a a a a

Thiabendazole* (positive control) 96.4 ± 0.47C 96.4 ± 0.47D 96.4 ± 0.47D 96.4 ± 0.47C

Capital letters compare mean in the columns and small letters compare mean in the lines. Different letters indicate signiﬁcantly different values (P < 0.05).

*PVPP concentration was 50 mg/ml and thiabendazole was 0.025 mg/ml.

tannins. Three repetitions were performed (Alonso-Díaz using SPSS 8.0 for Windows. For the LAEA, the results

et al., 2008). were analyzed by Kruskal-Wallis using the statistical

program Graph Pad Prism 5.0. The results were expressed

2.5. Phytochemical analysis of decoctions as the percentage SEM, and differences were considered

signiﬁcant at P < 0.05.

Phytochemical screening to characterize the major

classes of secondary metabolites present in the decoctions

3. Results

was performed according to the methodology proposed by

Matos (2009). The chemical characterization was based on

The average efﬁcacy of the decoctions to inhibit hatch-

the addition of speciﬁc reagents to decoction aliquots and

ing is shown in Table 1. At a concentration of 2.5 mg/ml,

observing the changes in solution color or precipitate for-

DTm and DMv showed efﬁcacies of 96.8and 97.6%, which

mation. The following experiments were performed: the

was not statistically different from the anthelmintic thi-

identiﬁcation of phenolic compounds (precipitation reac-

abendazole (P > 0.05). The EC50 values were 0.96 mg/ml for

tion with ferric chloride), the naphthoquinone reaction

DAz, 0.66 mg/ml for DTm and 0.5 mg/ml for DMv. DLc was

(acid/base), the characterization of ﬂavonoids (cyanidin

not effective at any concentration tested. DMv produced a

reaction and sulfuric acid), the presence of triterpenes and

more pronounced inhibitory effect on H. contortus larvae

steroids (Liebermann-Burchard reaction), alkaloids (pre-

inhibition hatching as compared with DAz and DTm.

cipitation reactions with Dragendorff and Mayer reagents)

Figs. 1–4 show the results of the larval artiﬁcial

and the characterization of saponins (Lieberman-Buchard

exsheathment assay. In the negative control, 97% of L3 were

reaction and the rate of spume).

exsheathed 60 min after contact with sodium hypochlorite

The content of total phenol was determined using the

solution. However, 3 h of treatment with the decoc-

Folin-Ciocalteu spectrophotometric method (Bonoli et al.,

tions blocked larval exsheathment. The addition of PVPP

2004). Brieﬂy, 100 ␮l of each extract solution (150 ppm)

to the decoctions reversed the inhibitory effect on the

was shaken for 1 min with 500 ␮l of Folin-Ciocalteu

larval exsheathment process: 60 min after contact with

reagent. Then, 2 ml of 15% sodium carbonate (Na2CO3)

sodium hypochlorite solution, the percentages of larvae

was added and the mixture was shaken again for 30 s. The

exsheathed were 95.6, 96.3, 99.6% and 100% for PVPP-

solution was adjusted to 10 ml by adding distilled water.

added DLc, DAz, DMv and DTm treatment, respectively.

After 2 h, measurements were performed on a spectropho-

Phytochemical screening showed the presence of con-

tometer at 750 nm. The readings, with three replicates per

densed tannins and ﬂavonoids in all decoctions. In the DLc,

sample, were performed with a negative control. The total

saponins were also present. The total phenols of the DLc,

phenol content was assessed by plotting the gallic acid

DAz, DMv and DTm were 87.5, 116.2, 117.7 and 45.2 mg/g

calibration curve. For the total tannin content, 100 mg/ml

(gallic acid equivalent), respectively and the total tannins

PVPP was used to isolate these metabolites from extract.

were 69.4, 107.1, 87.5 and 34.6 mg/g (gallic acid equiva-

Then total tannins were measured as the difference of

lent), respectively.

total phenol before and after treatment with PVPP (Oliveira

et al., 2011). The results for total phenols and tannins were

expressed as mg/g (equivalent to gallic acid). 4. Discussion

2.6. Statistical analysis The problem of anthelmintic resistance in addition

to the growing concern regarding the presence of drug

The results of EHT were expressed as the mean percent residues in animal products has led to an increased inter-

efﬁcacy of egg hatching inhibition ± standard error mean. est in the use of phytotherapeutics (Githiori et al., 2006).

The analysis was performed using ANOVA and compared In vitro tests with free-living stages of H. contortus have

by Tukey’s test (P < 0.05) using the Graph Pad Prism 5.0 been used for the preliminary assessment or screening of

program. The effective concentration to inhibit egg hatch- new plants with anthelmintic activity (Asase et al., 2005;

ing by 50% (EC50) was determined by the probit method Camurc¸ a-Vasconcelos et al., 2005).

I.T.F. Macedo et al. / Veterinary Parasitology 190 (2012) 504–509 507

120

100

20 Exsheathed larvae (%) 0 0 10 20 30 40 50 60

Time (min) (PBS) A. zerumbet Alpinia + PVPP

␮

Fig. 1. Effect of 310 g/ml of Alpinia zerumbet decoction, with or without addition of PVPP, on the process of artiﬁcial in vitro exsheathment of Haemonchus

contortus third-stage larvae.

120

Plants were selected and evaluated based on informa-

tion about their traditional use against helminths. The use 100

of decoctions to extract active ingredients from plants

mimics popular methods because decoctions have a greater

ease of preparation and are less toxic to manipulate (Schuch

et al., 2008). However, the technique of decoction may 40

alter many active substances by prolonged heating, and

are therefore considered to be a restriction on their use Exsheathed larvae (%)

(Falkenberg et al., 2000). This study veriﬁed the existence

0 10 20 30 40 50 60

of biologically active compounds with ovicidal effects on

H. contortus in DAz, DTm and DMv, even after heating for Time (min)

2 h. However, the low activity of DLc on the eggs can be (PBS) T. minuta T. minuta + PVPP

attributed to the lack of the ovicidal action of metabo-

␮

Fig. 3. Effect of 620 g/ml of Tagetes minuta decoction, with or with-

lites or the alteration of these compounds by heating. In

out addition of PVPP, on the process of artiﬁcial in vitro exsheathment

the study of the aqueous extract of Cucurbita moschata,

of Haemonchus contortus third-stage larvae.

Marie-Magdeleine et al. (2009) suggested that potentially

bioactive molecules could have been denatured, thereby

infectivity of pastures grazed by ruminants (Max, 2010).

inﬂuencing the anthelmintic activity.

DAz, DTm and DMv showed a dose-dependent inhibition of

The egg hatch test is an in vitro assay for assessing the

larvae hatching at lower concentrations compared to other

potential anthelmintic activities of natural products. Posi-

plants. The methanol extract of Annona squamosa had an

tive results on the inhibition of hatching eggs of a particular

EC50 of 3.8 mg/ml and the EC50 of ethyl acetate extracts

species of nematodes are considered to be an indicator

of Solanum torvum and Catharanthus roseus were 8.82 and

that the substance tested was effective against free-living

6.46 mg/ml, respectively (Kamaraj and Rahuman, 2010).

stages, infective larvae or adult worms in the host (Foster

The EC50 of the aqueous and hydro-alcoholic extracts of

et al., 2011). The capacity to reduce egg hatching could

Melia azedarach were 2.04 and 1.97 mg/ml, respectively

be of signiﬁcant epidemiological importance, and it could

(Kamaraj et al., 2010). The EC50 of the acetone extract of the

help to modulate the risk of parasitism by limiting the

stem of Myracrodruon urundeuva was 2.44 mg/ml (Oliveira

120 120

100 100

80 80

60 60

40 40

20 20 Exsheathed larvae (%) Exsheathed larvae larvae (%) Exsheathed

0 0

0 10 20 30 40 50 60 0 10 20 30 40 50 60

Time (min) Time (min)

(PBS) M. villosa M. villosa + PVPP (PBS) L. camara L. camara + PVPP

␮

Fig. 2. Effect of 310 g/ml of Mentha villosa, decoction, with or with- Fig. 4. Effect of 620 ␮g/ml of Lantana camara decoction, with or with-

out addition of PVPP, on the process of artiﬁcial in vitro exsheathment out addition of PVPP, on the process of artiﬁcial in vitro exsheathment of

of Haemonchus contortus third-stage larvae. Haemonchus contortus third-stage larvae.

508 I.T.F. Macedo et al. / Veterinary Parasitology 190 (2012) 504–509

et al., 2011). TheEC50 of the aqueous methanol extracts of A. zerumbetand M. villosa decoctions were the most potent

Musa paradisiaca and Trianthema portulacastrum were 2.13 at inducing exsheathment at a lower concentration.

and 2.41 mg/ml, respectively (Hussain et al., 2011). The results of the in vitro study support the traditional

Another in vitro test used to evaluate the effects of the use of A. zerumbet, T. minuta and M. villosa against nema-

decoctions was the LAEA. The L3 exsheathment represents todes. More research is needed to isolate and structurally

the transition from the free-living to the parasitic phase, identify the active compounds and to evaluate the toxicity

and is essential in the life cycle of nematodes. It has been and in vivo effects of these decoctions.

reported that tannins interrupt the process of exsheath-

ment, thus preventing the establishment of infective larvae Acknowledgements

in the host and consequently the infection (Brunet et al.,

␮

2007). The results demonstrated that 310 g/ml DAz or

This work received ﬁnancial support from the Conselho

␮

DMv and 620 g/ml DLc or DTm inhibited the process of

Nacional de Desenvolvimento Cientíﬁco e Tecnológico

H. contortus larvae exsheathment, showing superior efﬁ-

(CNPq) and Fundac¸ ão Cearense de Apoio ao Desenvolvi-

cacy as compared to 1000 ␮g/ml Jatropha curcas extract

mento Cientíﬁco e Tecnológico. Dr. Bevilaqua has a grant

(Monteiro et al., 2011).

from CNPq.

The biological effects of plants are mainly attributed

to the presence of secondary metabolites. The investi- References

gation of chemical components from natural products is

of fundamental importance for the development of new Ademola, I.O., Eloff, J.N., 2010. In vitro anthelmintic activity of Combretum

molle (R Br. ex G. Don) (Combretaceae) against Haemonchus contortus

anthelmintic drugs (Assis et al., 2003). In this study, phy-

ova and larvae. Vet. Parasitol. 169, 198–203.

tochemical tests revealed the presence of metabolites that

Albuquerque, U.P., Medeiros, P.M., Almeida, A.L., Monteiro, J.M., Neto,

may be responsible for anthelmintic activity. Among these E.M.F.L., Melo, J.G., Santos, J.P., 2007. Medicinal plants of the caatinga

components, saponins (Eguale et al., 2007; Ademola and (semi-arid) vegetation of NE Brazil: a quantitative approach. J.

Ethnopharmacol. 114, 325–354.

Eloff, 2010), ﬂavonoids (Lee et al., 2008) and tannins

Almeida, E.R. (Ed.), 1993. Plantas medicinais brasileiras – conhecimentos

(Oliveira et al., 2011) were detected.

populares e cientíﬁcos. Hemus, São Paulo, p. 341.

The role of condensed tannins in many biological pro- Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Aguilar-

Caballero, A.J., Hoste, H., 2008. In vitro larval migration and kinetics of

cesses related to different nematode stages have been

exsheathment of Haemonchus contortus larvae exposed to four tropical

conﬁrmed by in vitro studies (Hounzangbe-Adote et al.,

tanniniferous plant extracts. Vet. Parasitol. 153, 313–319.

2005). The mechanisms by which condensed tannins neu- Amarante, A.F.T., 2004. Controle integrado de helmintos de bovinos e ovi-

nos. Rev. Bras. Parasitol. Vet. 13, 68–71.

tralize the parasites can vary, depending on the nature and

Asase, A., Oteng-Yeboah, A.A., Odamtten, G.T., Simmonds, M.S.J., 2005.

composition of the different forage species (Brunet and

Ethnobotanical study of some Ghanaian anti-malarial plants. J.

Hoste, 2006; Brunet et al., 2007). Ethnopharmacol. 99, 273–279.

Assis, L.M., Bevilaqua, C.M.L., Morais, S.M., Vieira, L.S., Costa, C.T.C., Souza,

PVPP was used to demonstrate the effect of tannins

J.A.L., 2003. Ovicidal and larvicidal activity in vitro of Spigelia anthelmia

on L1 hatching and L3 exsheathment. In the egg hatch

Linn. extracts on Haemonchus contortus. Vet. Parasitol. 117, 43–49.

test, the anthelmintic efﬁcacy of the decoctions was not Athanasiadou, S., Kyriazakis, I., Jackson, F., Coop, R.L., 2001. Direct

anthelmintic effects of condensed tannins towards different gastroin-

totally abolished when they were incubated with PVPP,

testinal nematodes of sheep: in vitro and in vivo studies. Vet. Parasitol.

demonstrating that the tannins are not solely responsible

99, 205–219.

for the ovicidal activity, and other compounds present in Bahuaud, D., Martinez-Ortiz, M.C., Chauveau, S., Prevot, F., Torres-Acosta,

the decoctions may be acting alone or in synergy with the F., Fouraste, I., Hoste, H., 2006. Effects of four tanniferous plant extracts

on the in vitro exsheathment of third-stage larvae of parasitic nema-

tannins (Athanasiadou et al., 2001; Molan et al., 2003). This

todes. Parasitology 132, 545–554.

fact has been previously reported for the J. curcas extract

Bonoli, M., Verardo, V., Marconi, E., Caboni, M.F., 2004. Antioxidant

(Monteiro et al., 2011). phenols in barley (Hordeumvulgare L.) ﬂour: comparative spectropho-

tometric study among extraction methods of free and bound phenolic

In the larval exsheathment assay, the results observed

compounds. J. Agric. Food. Chem. 52, 5195–5200.

after PVPP addition showed that the tannins in the decoc-

Brunet, S., Hoste, H., 2006. Monomers of condensed tannins affect the lar-

tions are probably involved in the inhibitory effect on larval val exsheathment of parasitic nematodes of ruminants. J. Agric. Food

Chem. 54, 7481–7487.

exsheathment. Quantiﬁcation of tannins showed that DAz

Brunet, S., Aufrere, J., El Babili, F., Fouraste, H., Hoste, H., 2007. The kinetics

and DMv have higher levels of this metabolite, justifying its

of exsheathment of infective nematode larvae is disturbed in presence

higher effectiveness. The mechanism of action of tannins on of tannin-rich plant (sainfoin) both in vitro and in vivo. Parasitology

134, 1253–1262.

L3 remains largely unidentiﬁed, although many data sup-

Brunet, S., Jackson, F., Hoste, H., 2008. Effects of sainfoin (Onobrychis vici-

port the hypothesis of a direct effect (Brunet et al., 2008a).

ifolia) extract and monomers of condensed tannins on the association

In vitro results have shown that condensed tannins inter- of abomasal nematode larvae with fundic explants. Int. J. Parasitol. 38,

783–790.

fere with the two steps of the larval establishment, i.e., the

Brunet, S., Martinez-Ortiz de Montellano, C., Torres-Acosta, J.F.J., Sandoval-

exsheathment (Monteiro et al., 2011; Oliveira et al., 2011)

Castro, C.A., Aguilar-Caballero, A.J., Capetillo-Leal, C., Hoste, H., 2008a.

and the mucosal penetration (Brunet et al., 2008). The mode Effect of the consumption of Lysilomala tisilliquum on the larval estab-

of action of tannins on larval exsheathment may involve lishment of parasitic nematodes in goats. Vet. Parasitol. 157, 81–88.

Camurc¸ a-Vasconcelos, A.L.F., Morais, S.M., Santos, L.F.L., Rocha, M.F.G.,

the ability of tannins to bind to amino acids present in the

Bevilaqua, C.M.L., 2005. Validac¸ ão de plantas medicinais com ativi-

sheath, or to inactivate enzymes involved in this process

dade anti-helmíntica. Rev. Bras. Plantas Med. 7, 97–106.

and, consequently, affect the loss of sheath (Hoste et al., Coles, G.C., Bauer, C., Borgsteede, F.H.M., Geerts, S., Klei, T.R., Taylor,

2006). M.A., Waller, P.J., 1992. World Association for the Advancement of

veterinary parasitology (W.A.A.V.P.) methods for the detection of

Based on the EC , the most potent decoction was that of

50 anthelmintic resistance in nematodes of veterinary importance. Vet.

M. villosa. Based on the inhibition of larval exsheathment, Parasitol. 44, 35–44.

I.T.F. Macedo et al. / Veterinary Parasitology 190 (2012) 504–509 509

Costa, C.T.C., Morais, S.M., Bevilaqua, C.M.L., Souza, M.M.C., Leite, F.K.A., Marie-Magdeleine, C., Hoste, H., Mahieu, M., Varo, H., Archimede, H., 2009.

2002. Efeito ovicida de extratos de sementes de Mangifera indica L. In vitro effects of Cucurbita moschata seed extracts on Haemonchus

sobre Haemonchus contortus. Rev. Bras. Parasitol. Vet. 11, 57–60. contortus. Vet. Parasitol. 161, 99–105.

Eguale, T., Tilahun, G., Debella, A., Feleke, A., Makonnen, E., 2007. Matos, F.J.A. (Ed.), 2009. Introduc¸ ão à ﬁtoquímica experimental. UFC, For-

In vitro and in vivo anthelmintic activity of crude extracts of Corian- taleza, p. 45.

drum sativum against Haemonchus contortus. J. Ethnopharmacol. 110, Max, R.A., 2010. Effect of repeated wattle tannin drenches on worm

428–433. burdens, faecal egg counts and egg hatchability during naturally

Falkenberg, M.B., Santos, R.I., Simões, C.M.O., 2000. Introduc¸ ão à análise acquired nematode infections in sheep and goats. Vet. Parasitol. 169,

ﬁtoquímica. In: Simões, C.M.O., Schenkel, E.P., Gosmann, G., Mello, 138–143.

J.C.P., Mentz, L.A., Petrovick, P.R. (Eds.), Farmacognosia: da planta ao Melo, A.C.F.L., Reis, I.F., Bevilaqua, C.M.L., Vieira, L.S., Echevarria, F.A.M.,

medicamento. Universidade/UFRGS, Porto Alegre, pp. 163–179. Melo, L.M., 2003. Nematóides resistentes a anti-helmíntico em reban-

Foster, J.G., Cassida, K.A., Turner, K.E., 2011. In vitro analysis of the hos de ovinos e caprinos do estado do Ceará. Brasil. Cienc. Rural 37,

anthelmintic activity of forage chicory (Cichoriumintybus L.) sesquiter- 339–344.

pene lactones against a predominantly Haemonchus contortus egg Molan, A.L., Meagher, L.P., Spencer, P.A., Sivakumaran, S., 2003. Effect of

population. Vet. Parasitol. 180, 298–306. ﬂavan-3-ols on in vitro egg hatching, larval development and viability

Githiori, J.B., Athanasiadou, S., Thamsborg, S.M., 2006. Use of plants in of infective larvae of Trichostrongylus colubriformis. Int. J. Parasitol. 33,

novel approaches for control of gastrointestinal helminths in livestock 1691–1698.

with emphasis on small ruminants. Vet. Parasitol. 139, 308–320. Monteiro, M.V.B., Bevilaqua, C.M.L., Morais, S.M., Machado, L.K.A.,

Hammond, J.A., Fieding, D., Bishop, S.C., 1997. Prospects for plant Camurc¸ a-Vasconcelos, A.L.F., Campello, C.C., Ribeiro, W.L.C., Mesquita,

anthelmintics in tropical veterinary medicine. Vet. Res. Commun. 2, M.A., 2011. Anthelmintic activity of Jatropha curcas L. seeds on

213–228. Haemonchus contortus. Vet. Parasitol. 182, 259–263.

Hoste, H., Jackson, F., Athanasiadou, S., Thamsborg, S.M., Hoskin, S.O., 2006. Nunes, A.P., Oliveira, A.C., Berne, M.E.A., Borba, M.F.S., Echevarria, F.,

The effects of tannin-rich plants on parasitic nematodes in ruminants. Vaz, C.M., 2007. Estudo da variabilidade genética de resistência a

Trends Parasitol. 22, 253–261. nematódeos gastrintestinais em ovinos da rac¸ a Corriedale com mar-

Hounzangbe-Adote, M.S., Paolini, V., Fouraste, I., Moutairou, K., Hoste, H., cadores RAPD. R. Bras. Agrociência 3, 25–33.

2005. In vitro effects of four tropical plants on three life-cycle stages Oliveira, L.M.B., Bevilaqua, C.M.L., Macedo, I.T.F., Morais, S.M., Machado,

of the parasitic nematode, Haemonchus contortus. Res. Vet. Sci. 78, L.K.A, Campello, C.C., Mesquita, M.A., 2011. Effects of Myracro-

155–160. druon urundeuva extracts on egg hatching and larval exsheath-

Hubert, J., Kerboeuf, D., 1992. Amicrolarval development assay for the ment of Haemonchus contortus. Parasitol. Res. 109, 893–898,

detection of anthelmintic resistance in sheep nematodes. Vet. Rec. http://dx.doi.org/10.1007/s00436-011-2331-6.

151, 442–446. Rochfort, S., Parker, A.J., Dunshea, F.R., 2008. Plant bioactives for ruminant

Hussain, A., Khan, M.N., Iqbal, Z., Sajid, M.S., Khan, M.K., 2011. Anthelmintic health and productivity. Phytochemistry 69, 299–322.

activity of Trianthemaportulacastrum L. and Musaparadisiaca L. Schuch, L.F.D., Wiest, J.M., Garcia, E.N., Prestes, L.S., Schramm, R.C., Coim-

against gastrointestinal nematodes of sheep. Vet. Parasitol. 179, bra, H., Meireles, M.C.A., 2008. Atividade antifúngica de extratos de

92–99. plantas utilizados por agricultores familiares como antimicrobiano.

Kamaraj, C., Rahuman, A.A., 2010. Efﬁcacy of anthelmintic properties of Acta Scient. Vet. 36, 267–271.

medicinal plant extracts against Haemonchus contortus. Res. Vet. Sci. Scudeller, V.V., Veiga, J.B., Araújo-Jorge, L.H., 2009. Etnoconhecimento de

91, 400–404. plantas de uso medicinal nas comunidades São João do Tupé e Central

Kamaraj, C., Rahuman, A.A., Bagavan, A., Mohamed, M.J., Elango, G., Rajaku- (Reserva de Desenvolvimento Sustentável do Tupé). In: Santos-Silva,

mar, G., Zahir, A.A., Santhoshkumar, T., Marimuthu, S., 2010. Ovicidal E.N., Scudeller, V.V. (Eds.), Biotupé: Meio Físico, Diversidade Biológica

and larvicidal activity of crude extracts of Melia azedarach against e Sociocultural do Baixo Rio Negro,Amazônia Central. UEA Edic¸ ões,

Haemonchus contortus (Strongylida). Parasitol. Res. 106, 1071–1077, Manaus, pp. 185–199.

http://dx.doi.org/10.1007/s00436-010-1750-0. Ueno, H., Gonc¸ alves, P.C., 1998. Manual para diagnóstico das helmintoses

Lee, Y.U., Kawasaki, I., Lim, Y., Oh, W.S., Paik, Y.K., Shim, Y.H., 2008. de ruminantes. JIICA, Tokyo, Japan, pp. 143.

Inhibition of developmental processes by ﬂavonesin Caenorhabditis Waller, P.J., 2006. From discovery to development: current industry per-

elegans and its application to the pinewood nematode, Bursaphe- spectives for the development of novel methods of helminth control

lenchus xylophilus. Mol. Cells 26, 171–174. in livestock. Vet. Parasitol. 139, 1–14.