In vitro anthelmintic activity and safety of different plant species against the ovine gastrointestinal nematode Teladorsagia circumcincta
Graphical abstract
Introduction
Parasitic infections caused by gastrointestinal nematodes (GINs) are a worldwide health problem in grazing farm animals, mainly ruminants. These infections cause lack of appetite, diarrhea, anemia and, in severe cases, death (Athanasiadou and Kyriazakis, 2004). The usual mode of controlling GIN infections in ruminants is the administration of anthelmintic drugs. However, the misuse of these drugs has caused a rapid development of anthelmintic resistance in worm populations due to a rise in the frequency of resistant alleles (Martínez-Valladares et al., 2012a, Martínez-Valladares et al., 2012b). Moreover, the administration of these drugs leads to the presence of chemical residues in meat and milk, resulting in withdrawal periods that produce economic losses and, at the same time, increasing the risk of environmental contamination. These disadvantages have stimulated the search for alternative control methods, such as the use of new compounds with anthelmintic properties like some traditional medicinal plants.
In recent years many authors have reviewed the anthelmintic properties of different plants or extracts collected around the world (Acharya et al., 2014; Adamu et al., 2013; Eguale et al., 2007a, Eguale et al., 2007b; Kozan et al., 2006; Pessoa et al., 2002). Among many studies, one example is the study carried out by Lone et al. (2012) who found a significant anthelmintic activity, in vitro and in vivo, of aqueous and methanolic extracts of Euphorbia helioscopia against mixed GIN infections in sheep.
It is believed that the anthelmintic activity in plants is related to the amount of condensed tannins and/or others secondary metabolites such as flavonoids, steroids, terpenoids, alkaloids, saponins, etc. (Hoste et al., 2006, Hoste et al., 2012; Marie-Magdeleine et al., 2010; Vargas-Magaña et al., 2014). However, the amount of these compounds in the plant depends on the season when plant is collected, its geographical origin and phenological stage, among other factors (Manolaraki, 2011; Novobilsky et al., 2013). The importance of tannins is due to the fact that some studies in ruminants suggested that parasitised sheep and red deer grazing on forages high in condensed tannins had lower faecal egg counts and worm burdens compared with animals grazing on forages low in condensed tannins (Niezen et al., 1995; Hoskin et al., 1999). Later, Alonso-Díaz et al. (2008a), b) reported that four tropical tanniferous plants extracts interfered with the process of third stage larvae (L3) exsheathment of the GINs Haemonchus contortus and Trichostrongylus colubriformis, and also reduced the L3 migration of T. colubriformis. Moreover, Molan and Faraj (2010) observed that condensed tannins disrupted larval development of on in vitro assays with T. colubriformis and Teladorsagia circumcincta.
In this context, the present study was conducted to evaluate the in vitro anthelmintic activity by means of the Egg Hatch Assay (EHA) as well as the safety of aqueous and methanolic extracts obtained from 9 plants using the GIN T. circumcincta as model. As previously described in other studies (Acharya et al., 2014; Chan-Pérez et al., 2016; Vargas-Magaña et al., 2014), the EHA was used to determine the ovicidal activity of plant extracts; this activity is calculated according to the percentage of dead eggs after the exposure to the extract for 48 h. Also, the possible association of this anthelmintic activity with the total amount of phenols and tannins present in the extracts was determined.
Section snippets
Plant collection and extraction
A total of 9 plant species were selected for the current study based on their ethnomedicinal use in Spain, biological properties shown in previous studies and preliminary evidences observed in another study carried out by us but with different extract concentrations (unpublished data) (Takechi et al., 1985; Kozan et al., 2006; Meng et al., 2009; Abbasipour et al., 2011; Zhou and Qu, 2011). Among them, the aerial parts (a mixture of leaves, flowers and stems) of 7 plants were collected from
Extract yields
The yields of aqueous and methanolic extracts (w/w) are shown in Table 2, ranging from 7.2% to 27.3%. The highest yield was observed for the aqueous extract of S. irio and the lowest one was shown for the methanolic extract of the same plant. After the evaporation of the methanolic solution of E. cardamomum an oily extract was obtained, therefore this extract was tested like an essential oil.
Solubility of the extracts
With the aim to solubilize the extracts to prepare stock solutions for the EHA, different diluents,
Discussion
The main objective of this study was to assess the possible anthelmintic activity and safety of aqueous and methanolic extracts obtained from aerial parts of 7 plants and 2 seeds. With this purpose the inhibition of egg hatching of T. circumcincta, a GIN infecting sheep, was studied by means of the EHA. According to Azmir et al. (2013), water is a good solvent for many bioactive compounds from plants such as anthocyanins, terpenoids, saponins and tannins, and methanol is a good solvent for
Conclusions
According to the results shown in the current study, the methanolic extract of E. cardamomum and the aqueous extracts of S. irio, with EC50 values of 0.37% and 2.41 mg/ml respectively, could be candidates for further isolation in vivo assays since their SI were equal or higher than 1. Moreover, due to its low toxicity with a SI higher than 1.5, it could be worthwhile to test in vivo the aqueous extract of J. polyanthum, although its EC50 is slightly higher than the threshold proposed to achieve
Acknowledgements
This study has been funded by the national project INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria: RTA2013-00064-C02-02), Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad: AGL 2016-79813-C2-1R), the European Regional Development Fund (Fondos Feder) and the Spanish “Ramón y Cajal” Programme, Ministry of Economy and Competitiveness (MMV, RYC-2015-18368).
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