Oxalis Tetraphylla (Oxalidaceae) Hydroalcoholic Extract Against Haemonchus Contortus in Lambs
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Journal of Helminthology (2018) 92, 309–316 doi:10.1017/S0022149X17000499 © Cambridge University Press 2017 In vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla (Oxalidaceae) hydroalcoholic extract against Haemonchus contortus in lambs B.J. González-Cruz1, M. Rodríguez-Labastida1, M. González-Cortázar2, A. Zamilpa2, M.E. López-Arellano1, L. Aguilar-Marcelino1, R. González-Garduño3, J.F.J. Torres-Acosta4, A. Olmedo-Juárez1 and P. Mendoza-de Gives1* 1Departamento de Helmintologia, Centro Nacional de Parasitología Veterinaria, INIFAP, Boulevard Paseo Cuaunahuac No. 8534, Col. Progreso, Jiutepec, Morelos, México, CP 62550: 2Centro de Investigación Biomédica del Sur (CIBIS-IMSS), Argentina No. 1 Centro, 62790 Xochitepec, Morelos, México: 3Facultad de Medicina Veterinaria y Zootecnia, Centro Regional Universitario del Sureste, Km. 7 Carretera Teapa-Vicente Guerrero, Ranchería San José Puyacatengo, Teapa, Tabasco, México: 4Universidad Autónoma de Yucatán, Km 15.5 Carretera Mérida-Xmatkuil, Mérida, Yucatán, México CP 97000 (Received 9 February 2017; Accepted 10 May 2017; First published online 9 June 2017) Abstract The in vitro larvicidal and in vivo anthelmintic effects of Oxalis tetraphylla hydroalcoholic extract (HE), against Haemonchus contortus in experimentally infected lambs, were assessed. We used a microtitration plate method, compris- ing the following two stages. Stage 1: 20 μl of water containing 200 sheathed H. contortus infective larvae (ShHcl) were deposited in every well of three series; then, the series 2 and 3 wells were treated with 80 μl 1% ivermectin and O. tetra- phylla HE at 20 mg/ml, respectively. Stage 2: the same procedure was performed replacing the ShHcl with exsheathed larvae (ExShHcl). Evaluations were performed after 24 and 48 h. The total numbers of dead and live larvae were counted. A second experiment evaluated the reduction in nematode egg populations in the faeces of lambs treated orally with the O. tetraphylla HE. The 27 lambs used were divided into Groups 1, 2 and 3 (n = 9), which were ad- ministered water (positive control), levamisole 1 M (7.5 mg/kg body weight (BW), as a unique dose) and O. tetraphylla HE (20 mg/kg BW), respectively. The plant HE was administered daily for 8 days. The in vitro assay showed 80.9% and 86.5% larval mortality of ShHcl after 24 and 48 h, respectively, while the corresponding mortality values for ExShHcl were 97 and 99%, respect- ively. The in vivo assay showed variability in the eggs/gram of faeces (epg) values; however, at the end of the trial, the average reduction in the epg values of the O. tetraphylla HE group was 45.6% (P < 0.05). Oxalis tetraphylla HE contains compounds that belong to the flavonol group with anthelmintic activity. *Fax: + 52 (777) 3192848 E-mail: [email protected] Downloaded from https://www.cambridge.org/core. IP address: 170.106.202.126, on 25 Sep 2021 at 06:25:35, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X17000499 310 B.J. González-Cruz et al. Introduction in vivo anthelmintic activity of plant extracts involves slaughtering the animal and then counting the adult Sheep haemonchosis is considered to be one of the main worms recovered from the digestive tract. However, this health problems affecting sheep flocks worldwide method requires the euthanization of numerous animals, (Fiaz-Qamar et al., 2011). Infected animals show clinical which is very expensive. Another technique that allows signs such as anorexia, weight loss, diarrhoea, submaxil- the identification of potential anthelmintic compounds is lary oedema, slow growth, malnutrition (Prakash & based on counting the number of eggs/gram of faeces Bano, 2010) and even death of young animals (Garedaghi (epg) before and after treatments (Irum et al., 2015). The et al., 2013). Haemonchosis and other gastrointestinal para- method is simple, inexpensive and avoids the euthaniza- sitic nematodes (GIPN) have been controlled mainly with tion of animals. The present study was designed to assess chemical anthelmintic drugs that are administered as a con- both the in vitro nematocidal activity against Haemonchus tinuous treatment strategy for de-worming animals. contortus infective larvae (L3) and the in vivo effect of oral However, this system has led to the development of anthel- administration of O. tetraphylla hydroalcoholic extract mintic resistance (AR) (Torres-Acosta et al., 2012) as well as (HE) on the faecal egg count in lambs experimentally in- health risks due to the possible persistence of chemical resi- fected with H. contortus. dues in animal products. The use of ethnoveterinary or traditional medicines in veterinary practice has gained the attention of numerous researchers worldwide, and Materials and methods some plants have been reported to be excellent candidates Plant material for controlling animal parasitosis caused by nematodes (Akerreta et al., 2010; Bharati & Sharma, 2012; Hassan Wild specimens of O. tetraphylla were collected from a et al., 2014; Saha et al., 2014). Plants such as Trifolium repens forest at the top of the ‘Cerro light’ mountain (mountain and Vernona anthelma are currently being investigated as of light) in the village of San Juan Tlacotenco, Tepoztlán possible sources of agents to control GIPN in ruminants Municipality, Morelos, Mexico, in January 2012. A vou- (Heckendorn et al., 2007; Alawa et al., 2010). Oxalis cher specimen of the plant material was deposited at the tetraphylla is a bulbous plant from Mexico and Guatemala herbarium of the National Institute of Anthropology that belongs to the Oxalidaceae family, which has approxi- and History of Mexico, Cuernavaca City, Morelos State, mately 900 species (Burger, 1991). It is widely distributed in Mexico (registration number: 2058). A biologist, South America, Mexico and Africa (Loudon, 2009). It is Margarita Aviléz, established the taxonomy of the plant traditionally called ‘Trebol de la Buena Suerte’ (good luck specimen. clover) and possesses four triangular green leaves with pur- ple colour diffused at the centre of the leaves (Brickell & Extract preparation Zuk, 1997)(fig. 1). Although this plant has been used for human consumption, it is mostly used for cattle grazing Five kilograms of the wild leaves and stems were dried (Romero, 2000). The most reliable assay for assessing the and then extracted three times by maceration in an Fig. 1. Leaves of the leguminous plant Oxalis tetraphylla collected from the top of the Woodland Mountain at San Juan Tlacotenco village, Tepoztlán, Morelos State, Mexico. Downloaded from https://www.cambridge.org/core. IP address: 170.106.202.126, on 25 Sep 2021 at 06:25:35, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0022149X17000499 Effects of Oxalis tetraphylla extract against H. contortus in lambs 311 ethanol–water mixture (60:40, v/v, 15 litres; Merck, gradients of a 40% saccharose solution, rinsed and then Darmstadt, Germany) for 24 h at room temperature (18– suspended in sterile water. 25°C). This maceration process was described previously by De Jesús Gabino et al.(2010). The HE of each plant ma- terial sample was concentrated and completely dried Experiment 1: in vitro assessment of effects of O. tetraphylla using a rotatory evaporator (Heidolph Laborota 4000; HE against L3 Heidolph Instruments, Schwabach, Germany) under re- Experimental design duced pressure at 50–60°C to obtain 38.1 g of HE extract. Two experimental stages were used to assess the in vitro activity of the O. tetraphylla HE against sheathed and ex- sheathed L3. The larvae/extract treatment was carried Phytochemical analysis using thin layer chromatography out in 96-well plates. Three wells per treatment were con- To identify the chemical compounds in the O. tetraphylla sidered as three experimental units. The design of each ex- HE, we used a thin layer chromatography (TLC) technique perimental stage was similarly structured, and three series to analyse specific chemical reactions (Wagner et al., 1984). of three wells were treated as follows. Stage 1: first, 20 μlof We used a visualization agent (2-aminoethyl diphenylbor- water containing 200 sheathed L3 were placed in every inate in polyethylene glycol) to detect flavonoids, which well of the three series. Series 1 contained only larvae; were observed under ultraviolet (UV) light at a long wave- in series 2 and 3, 80 μl of 1% ivermectin or O. tetraphylla length (360 nm). A comparison of the TLC analysis of sam- HE at a concentration of 20 mg/ml were added and ples previously isolated indicated that the detected mixed. Stage 2 followed the same procedure as that compounds were β-sitosterol and stigmasterol sterols, used in Stage 1 except that exsheathed larvae were used. which are known to have widespread occurrence in the Two incubation periods were used (24 and 48 h post- plant kingdom. The compounds were revealed using the treatment). After incubation, ten 5-μl aliquots (considered Komarowski reagent (4-hydroxybenzaldehyde in sulphur- as n = 10 aliquots/well from three experimental units per ic acid). treatment) were placed on a slide and observed under a microscope at 4× and 10× magnifications. The total, dead and live larvae were counted. The criteria for identi- Phytochemical analysis using high-performance liquid fying the dead and live larvae were based on their mobil- chromatography ity/immobility; physical stimuli were applied to confirm The high-performance liquid chromatography (HPLC) the dead and live larvae. The proportions of dead and analysis was performed using a Waters 2695 separation live larvae were estimated for each series. The proportion module system equipped with a Waters 2996 photodiode of live larvae in series 1 (control, water) was considered as array detector and the Empower Chromatographic 100% and used for comparison. Ponder adjustments were Manager version 1 software (Waters, Milford, performed when necessary considering the number of lar- Connecticut, USA). The analysis was performed using a vae that died for reasons other than the treatments.