Veterinary Parasitology

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1 Veterinary Parasitology 19 (12) Contents lists available at SciVerse ScienceDirect Veterinary Parasitology jou rn al h om epa ge: In vitro activity of Lantana camara, Alpinia zerumbet, Mentha villosa and Tagetes minuta decoctions on Haemonchus contortus eggs and larvae Iara T.F. Macedo a, Claudia M.L. Bevilaqua a,, Lorena M.B. de Oliveira a, Ana L.F. Camurç a-vasconcelos a, Selene M. Morais a, Lyeghyna K.A. Machado b, Wesley L.C. Ribeiro a a Programa de Pós-Graduaç 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 Article history: Received 1 November 11 Received in revised form 22 June 12 Accepted 2 July 12 Keywords: Phytotherapy Anthelmintic Gastrointestinal nematodes Tannins a b s t r a c t The resistance of gastrointestinal nematodes to anthelmintics has increased the need to evaluate natural products that can replace or assist current strategies to control gastrointestinal nematodes. The objective of this study was to evaluate the effect of decoctions 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 concentrations of lyophilized decoctions (.31 to 1 mg/ml) were assessed using the egg hatch test (EHT). The decoctions were then tested in the larval artificial exsheathment assay. H. contortus third stage larvae (L3) were exposed to.31 mg/ml A. zerumbet and M. villosa decoctions and.62 mg/ml T. minuta and L. camara decoctions for 3 h and then exsheathment procedure at 1 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. 12 Elsevier B.V. Open access under the Elsevier OA license. 1. Introduction The diseases caused by gastrointestinal nematode parasites occupy a prominent place among the factors limiting sheep and goat production and are responsible for high economic losses due to delayed growth, weight loss, reduced Corresponding author at: Programa de Pós-graduaç ão em Ciências Veterinárias/FAVET/UECE, Av. Dede Brasil, 17, Campus do Itaperi, CEP , Fortaleza, Ceará, Brazil. Tel.: ; fax: address: claudiamlb@yahoo.com.br (C.M.L. Bevilaqua). food consumption, decreased milk production, low fertility and, in cases of massive infections, high mortality rates (Nunes et al., 7). The high prevalence associated with pathogenicity make the hematophagous nematode Haemonchus contortus the main parasite of small ruminants in Brazil (Amarante, 4). The control of parasitism is accomplished primarily through the use of commercial semi-synthetic and synthetic anthelmintics. However, these drugs are associated with drawbacks, the risk of residues in food and environmental contamination (Waller, 6). Moreover, misuse and indiscriminate treatment with synthetic drugs have allowed the rapid selection of resistant helminth populations (Melo et al., 3). Thus, it / 12 Elsevier B.V. Open access under the Elsevier OA license.

2 I.T.F. Macedo et al. / Veterinary Parasitology 19 (12) has become necessary to develop studies aimed at searching for complementary alternatives to traditional methods (Ademola and Eloff, 1). The use of plants with anthelmintic properties seems to be an effective alternative, both from the standpoint of parasite control and their low environmental impact (Hammond et al., 1997). The natural products are a mixture of components which act in synergy producing the anthelminthic effect, differing from the commercial drugs which have one molecule acting on the parasite when not combination formulation. Thus, the resistance is likely to develop more slowly in the natural product. Plant extracts have been used since antiquity as remedies for many diseases. Infusions and aqueous decoctions are the main methods of preparation used in traditional medicine (Scudeller et al., 9). Among the species of plant that have been documented as having medicinal effects against helminths are Lantana camara (Verbenaceae), Tagetes minuta (Asteraceae), Mentha villosa (Lamiaceae) (Albuquerque et al., 7) and Alpinia zerumbet (Zingiberaceae) (Almeida, 1993). Recent studies have suggested that plants containing condensed tannins may offer a promising alternative approach to control parasitism (Rochfort et al., 8). Condensed tannins are secondary plant metabolites that comprise the most widespread class in nature. The value and credibility of herbal medicines depend on the recognition of the healing properties of some plants. Thus, scientific experimentation is a crucial and obligatory step to prove the effectiveness of plants popularly used as anthelmintics (Githiori et al., 6). To assess the anthelmintic properties of plant extracts, in vitro testing can be used as a preliminary step to characterize the possible effects (Costa et al., 2). The objective of this study was to evaluate the effects of decoctions of L. camara (DLc), A. zerumbet (DAz), M. villosa (DMv) and T. minuta (DTm) on egg hatching and larval exsheathment of H. contortus. 2. Materials and methods 2.1. Decoction extraction The plants used were collected in the Horto of medicinal plants of the Universidade Federal do Ceará in plots exposed to environmental conditions in a tropical country in Fortaleza, State of Ceará, Brazil. All plants were authenticated and voucher specimens were deposited in the Herbarium Prisco Bezerra of the Universidade Federal do Ceará, under the numbers: L. camara- 46,17, A. zerumbet- 49,659, M. villosa- 596 and T. minuta- 49,676. For the production of the decoctions, fresh plant material was cut from aerial parts and completely submerged in distilled water at 1:1 (g/ml) and boiled for 2 h under heating at reflux with reconstitution of the evaporated volume. Subsequently, the solutions were filtered, frozen and lyophilized. The decoctions stored at 4 C until used. The decoctions were soluble in water Obtaining H. contortus eggs and larvae One 6-month old, male sheep, of mongrel breed, was kept in a metabolic cage and initially treated with three anthelmintics with different active ingredients (Fenbendazole, Panacur ; Levamisole, Ripercol, and Ivermectin, Ivomec )on alternate days to eliminate gastrointestinal nematodes. Five thousand infective larvae (L3) of H. contortus benzimidazole-resistant were orally inoculated 21 days after treatment. Subsequently fecal samples were collected to confirm the establishment of the experimental infection, using a modified McMaster technique, obtaining an egg count per gram of feces (epg) above,. To recover H. contortus eggs, 1 g of feces collected directly from the rectum of the experimentally infected sheep was processed according to the technique described by Hubert and Kerboeuf (1992). L3 were collected from feces according to Ueno and Gonç alves (1998) Egg hatch test (EHT) This test was performed based on the methodology described by Coles et al. (1992). To increase the solubility in aqueous medium, the decoctions were diluted in 3% Tween 8. An egg suspension (25 l) containing approximately 1 fresh eggs were incubated with 25 l decoctions at final concentrations of.31 1 mg/ml for 48 h at 25 C. Drops of Lugol were added, and the eggs and first stage larvae (L1) were counted under a microscope. This test had two controls: a negative control containing the diluent (Tween 8) and a positive control with.25 mg/ml thiabendazole (diluted with 3% dimethylsulfoxide-dmso). Three repetitions in different days with five replicates for each decoction concentration and for each control were performed. To evaluate the role of tannins on the anthelmintic effect, a specific inhibitor of tannins, polyvinyl polypyrrolidone (PVPP; Sigma ), was used. The PVPP binds to tannins inhibiting from exercising their effect, i.e., allowing the egg hatch. The highest concentrations of DAz, DTm and DMv were pre-incubated overnight with 5 mg/ml PVPP (Alonso-Díaz et al., 8) Larval artificial exsheathment assay (LAEA) This test was based on the methodology described by Bahuaud et al. (6). One thousand H. contortus L3 were incubated for 3 h in a solution containing decoction diluted in phosphate buffer solution (PBS) at concentrations of 31 g/ml (DAz and DMv) or 6 g/ml (DLc and DTm) or the negative control with 1 ml of PBS. Larvae were then washed and centrifuged three times in PBS and subjected to an artificial exsheathment process by contact with a solution of sodium hypo chloride (2% w/v) diluted 1:3 in PBS. The kinetics of larval exsheathment were monitored microscopically for 1 h to identify the exsheathed larvae at, 1,, 3, 4, 5 and 6 min. Six replicates were performed for each decoction and the control to examine the changes in the proportion of exsheathed larvae in relation to time. Each decoction concentration was pre-incubated overnight with 5 mg/ml PVPP to evaluate the role of

3 56 I.T.F. Macedo et al. / Veterinary Parasitology 19 (12) 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 ± 1.2A a 12.1 ± 1.77A a 24.7 ± 1.74A b ±.84AB a 18.5 ± 1.57A a 44.6 ± 4.1B b 62.7 ± 2.87B c ±.74AB a 74.6 ± 1.96B b 85.8 ± 2.4C c 9. ± 2.18CD c ± 1.13A a 82.7 ± 1.55C b 96.8 ±.77D c 97.6 ±.79C c ± 1.66AB a 97.5 ±.84D b 1 ±.D b ± 2.15B Pvpp* + decoction 82.6 ± 1.46C a 92.8 ± 2.4CD a 84.3 ± 4.5D a Tween 8 (3%) (negative control) 13. ±.77A a 14.3 ± 1.5A a 13.7 ± 1.84A a 11.2 ± 1.46E a Thiabendazole* (positive control) 96.4 ±.47C a 96.4 ±.47D a 96.4 ±.47D a 96.4 ±.47C a Capital letters compare mean in the columns and small letters compare mean in the lines. Different letters indicate significantly different values (P <.5). *PVPP concentration was 5 mg/ml and thiabendazole was.25 mg/ml. tannins. Three repetitions were performed (Alonso-Díaz et al., 8) Phytochemical analysis of decoctions Phytochemical screening to characterize the major classes of secondary metabolites present in the decoctions was performed according to the methodology proposed by Matos (9). The chemical characterization was based on the addition of specific reagents to decoction aliquots and observing the changes in solution color or precipitate formation. The following experiments were performed: the identification of phenolic compounds (precipitation reaction with ferric chloride), the naphthoquinone reaction (acid/base), the characterization of flavonoids (cyanidin reaction and sulfuric acid), the presence of triterpenes and steroids (Liebermann-Burchard reaction), alkaloids (precipitation reactions with Dragendorff and Mayer reagents) and the characterization of saponins (Lieberman-Buchard reaction and the rate of spume). The content of total phenol was determined using the Folin-Ciocalteu spectrophotometric method (Bonoli et al., 4). Briefly, 1 l of each extract solution (15 ppm) was shaken for 1 min with 5 l of Folin-Ciocalteu reagent. Then, 2 ml of 15% sodium carbonate (Na 2 CO3) was added and the mixture was shaken again for 3 s. The solution was adjusted to 1 ml by adding distilled water. After 2 h, measurements were performed on a spectrophotometer at 75 nm. The readings, with three replicates per sample, were performed with a negative control. The total phenol content was assessed by plotting the gallic acid calibration curve. For the total tannin content, 1 mg/ml PVPP was used to isolate these metabolites from extract. Then total tannins were measured as the difference of total phenol before and after treatment with PVPP (Oliveira et al., 11). The results for total phenols and tannins were expressed as mg/g (equivalent to gallic acid) Statistical analysis The results of EHT were expressed as the mean percent efficacy of egg hatching inhibition ± standard error mean. The analysis was performed using ANOVA and compared by Tukey s test (P <.5) using the Graph Pad Prism 5. program. The effective concentration to inhibit egg hatching by 5% (EC 5 ) was determined by the probit method using SPSS 8. for Windows. For the LAEA, the results were analyzed by Kruskal-Wallis using the statistical program Graph Pad Prism 5.. The results were expressed as the percentage ± SEM, and differences were considered significant at P < Results The average efficacy of the decoctions to inhibit hatching is shown in Table 1. At a concentration of 2.5 mg/ml, DTm and DMv showed efficacies of 96.8and 97.6%, which was not statistically different from the anthelmintic thiabendazole (P >.5). The EC 5 values were.96 mg/ml for DAz,.66 mg/ml for DTm and.5 mg/ml for DMv. DLc was not effective at any concentration tested. DMv produced a more pronounced inhibitory effect on H. contortus larvae inhibition hatching as compared with DAz and DTm. Figs. 1 4 show the results of the larval artificial exsheathment assay. In the negative control, 97% of L3 were exsheathed 6 min after contact with sodium hypochlorite solution. However, 3 h of treatment with the decoctions blocked larval exsheathment. The addition of PVPP to the decoctions reversed the inhibitory effect on the larval exsheathment process: 6 min after contact with sodium hypochlorite solution, the percentages of larvae exsheathed were 95.6, 96.3, 99.6% and 1% for PVPPadded DLc, DAz, DMv and DTm treatment, respectively. Phytochemical screening showed the presence of condensed tannins and flavonoids in all decoctions. In the DLc, saponins were also present. The total phenols of the DLc, DAz, DMv and DTm were 87.5, 116.2, and 45.2 mg/g (gallic acid equivalent), respectively and the total tannins were 69.4, 17.1, 87.5 and 34.6 mg/g (gallic acid equivalent), respectively. 4. Discussion The problem of anthelmintic resistance in addition to the growing concern regarding the presence of drug residues in animal products has led to an increased interest in the use of phytotherapeutics (Githiori et al., 6). In vitro tests with free-living stages of H. contortus have been used for the preliminary assessment or screening of new plants with anthelmintic activity (Asase et al., 5; Camurç a-vasconcelos et al., 5).

4 I.T.F. Macedo et al. / Veterinary Parasitology 19 (12) (PBS) A. zerumbet Alpinia + PVPP Fig. 1. Effect of 31 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. Plants were selected and evaluated based on information about their traditional use against helminths. The use 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., 8). However, the technique of decoction may alter many active substances by prolonged heating, and are therefore considered to be a restriction on their use (Falkenberg et al., ). This study verified the existence of biologically active compounds with ovicidal effects on H. contortus in DAz, DTm and DMv, even after heating for 2 h. However, the low activity of DLc on the eggs can be attributed to the lack of the ovicidal action of metabolites or the alteration of these compounds by heating. In the study of the aqueous extract of Cucurbita moschata, Marie-Magdeleine et al. (9) suggested that potentially bioactive molecules could have been denatured, thereby influencing the anthelmintic activity. The egg hatch test is an in vitro assay for assessing the potential anthelmintic activities of natural products. Positive results on the inhibition of hatching eggs of a particular species of nematodes are considered to be an indicator that the substance tested was effective against free-living stages, infective larvae or adult worms in the host (Foster et al., 11). The capacity to reduce egg hatching could be of significant epidemiological importance, and it could help to modulate the risk of parasitism by limiting the (PBS) T. minuta T. minuta + PVPP Fig. 3. Effect of 6 g/ml of Tagetes minuta decoction, with or without addition of PVPP, on the process of artificial in vitro exsheathment of Haemonchus contortus third-stage larvae. infectivity of pastures grazed by ruminants (Max, 1). DAz, DTm and DMv showed a dose-dependent inhibition of larvae hatching at lower concentrations compared to other plants. The methanol extract of Annona squamosa had an EC 5 of 3.8 mg/ml and the EC 5 of ethyl acetate extracts of Solanum torvum and Catharanthus roseus were 8.82 and 6.46 mg/ml, respectively (Kamaraj and Rahuman, 1). The EC 5 of the aqueous and hydro-alcoholic extracts of Melia azedarach were 2.4 and 1.97 mg/ml, respectively (Kamaraj et al., 1). The EC 5 of the acetone extract of the stem of Myracrodruon urundeuva was 2.44 mg/ml (Oliveira (PBS) M. villosa M. villosa + PVPP (PBS) L. camara L. camara + PVPP Fig. 2. Effect of 31 g/ml of Mentha villosa, decoction, with or without addition of PVPP, on the process of artificial in vitro exsheathment of Haemonchus contortus third-stage larvae. Fig. 4. Effect of 6 g/ml of Lantana camara decoction, with or without addition of PVPP, on the process of artificial in vitro exsheathment of Haemonchus contortus third-stage larvae.

5 58 I.T.F. Macedo et al. / Veterinary Parasitology 19 (12) et al., 11). TheEC 5 of the aqueous methanol extracts of Musa paradisiaca and Trianthema portulacastrum were 2.13 and 2.41 mg/ml, respectively (Hussain et al., 11). Another in vitro test used to evaluate the effects of the decoctions was the LAEA. The L3 exsheathment represents the transition from the free-living to the parasitic phase, and is essential in the life cycle of nematodes. It has been reported that tannins interrupt the process of exsheathment, thus preventing the establishment of infective larvae in the host and consequently the infection (Brunet et al., 7). The results demonstrated that 31 g/ml DAz or DMv and 6 g/ml DLc or DTm inhibited the process of H. contortus larvae exsheathment, showing superior efficacy as compared to 1 g/ml Jatropha curcas extract (Monteiro et al., 11). The biological effects of plants are mainly attributed to the presence of secondary metabolites. The investigation of chemical components from natural products is of fundamental importance for the development of new anthelmintic drugs (Assis et al., 3). In this study, phytochemical tests revealed the presence of metabolites that may be responsible for anthelmintic activity. Among these components, saponins (Eguale et al., 7; Ademola and Eloff, 1), flavonoids (Lee et al., 8) and tannins (Oliveira et al., 11) were detected. The role of condensed tannins in many biological processes related to different nematode stages have been confirmed by in vitro studies (Hounzangbe-Adote et al., 5). The mechanisms by which condensed tannins neutralize the parasites can vary, depending on the nature and composition of the different forage species (Brunet and Hoste, 6; Brunet et al., 7). PVPP was used to demonstrate the effect of tannins on L1 hatching and L3 exsheathment. In the egg hatch test, the anthelmintic efficacy of the decoctions was not totally abolished when they were incubated with PVPP, demonstrating that the tannins are not solely responsible for the ovicidal activity, and other compounds present in the decoctions may be acting alone or in synergy with the tannins (Athanasiadou et al., 1; Molan et al., 3). This fact has been previously reported for the J. curcas extract (Monteiro et al., 11). In the larval exsheathment assay, the results observed after PVPP addition showed that the tannins in the decoctions are probably involved in the inhibitory effect on larval exsheathment. Quantification of tannins showed that DAz and DMv have higher levels of this metabolite, justifying its higher effectiveness. The mechanism of action of tannins on L3 remains largely unidentified, although many data support the hypothesis of a direct effect (Brunet et al., 8a). In vitro results have shown that condensed tannins interfere with the two steps of the larval establishment, i.e., the exsheathment (Monteiro et al., 11; Oliveira et al., 11) and the mucosal penetration (Brunet et al., 8). The mode of action of tannins on larval exsheathment may involve the ability of tannins to bind to amino acids present in the sheath, or to inactivate enzymes involved in this process and, consequently, affect the loss of sheath (Hoste et al., 6). Based on the EC 5, the most potent decoction was that of M. villosa. Based on the inhibition of larval exsheathment, A. zerumbetand M. villosa decoctions were the most potent at inducing exsheathment at a lower concentration. The results of the in vitro study support the traditional use of A. zerumbet, T. minuta and M. villosa against nematodes. More research is needed to isolate and structurally identify the active compounds and to evaluate the toxicity and in vivo effects of these decoctions. Acknowledgements This work received financial support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundaç ão Cearense de Apoio ao Desenvolvimento Científico e Tecnológico. Dr. Bevilaqua has a grant from CNPq. References Ademola, I.O., Eloff, J.N., 1. In vitro anthelmintic activity of Combretum molle (R Br. ex G. Don) (Combretaceae) against Haemonchus contortus ova and larvae. Vet. Parasitol. 169, Albuquerque, U.P., Medeiros, P.M., Almeida, A.L., Monteiro, J.M., Neto, E.M.F.L., Melo, J.G., Santos, J.P., 7. Medicinal plants of the caatinga (semi-arid) vegetation of NE Brazil: a quantitative approach. J. Ethnopharmacol. 114, Almeida, E.R. (Ed.), Plantas medicinais brasileiras conhecimentos populares e científicos. Hemus, São Paulo, p Alonso-Díaz, M.A., Torres-Acosta, J.F.J., Sandoval-Castro, C.A., Aguilar- Caballero, A.J., Hoste, H., 8. 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