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In Vitro Activity of Lantana Camara, Alpinia Zerumbet, Mentha Villosa And 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 , a Wesley L.C. Ribeiro a Programa de Pós-Graduac¸ ão em Ciências Veterinárias, Universidade Estadual do Ceará, Brazil b 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. © 2012 Elsevier B.V. Open access under the Elsevier OA license. 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 0304-4017/ © 2012 Elsevier B.V. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.vetpar.2012.07.001 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.
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