title: Elucidation of Leucaena leucocephala anthelmintic-like phytochemicals and the ultrastructural damage generated to eggs of Cooperia spp.
abstract: Leucaena leucocephala is a tropical forage legume suggested as an alternative method to control gastrointestinal parasitism in ruminants. This study: (1) performed a bio-guided fractionation of an aqueous extract of L. leucocephala using the egg hatch assay (EHA) to identify the anthelmintic (AH)-like phytochemicals present in fresh leaves, and (2) assessed the ultrastructural damage to eggs of Cooperia spp. after incubation with the final fraction. Phytochemicals were isolated using silica gel columns and identified using high performance liquid chromatography and standards for comparison. The final fraction was evaluated using EHA at 0.06, 0.125, 0.250, 0.500 and 1.1 mg ml(-1). The lethal concentration to inhibit 50% of Cooperia spp. egg hatching (LC50) was calculated using a Probit analysis. Scanning and transmission electron microscopy revealed the ultrastructural changes present in Cooperia spp. eggs. Bio-guided isolation procedures led to the recognition of an active fraction (LlC1F3) mainly composed of quercetin (82.21%) and caffeic acid (13.42%) which inhibited 90.49 ± 2.8% of Cooperia spp. egg hatching (P<0.05), and an LC50 of 0.06 ± 0.14 mg ml(-1). Scanning electron microscopy (SEM) showed eggs exposed to the active fraction had an irregular external layer with small projections and ruptures of lateral eggshell walls. Transmission electron microscopy (TEM) showed changes to Cooperia spp. eggs in electro-density, including the thickness of the eggshell layers and fractures after incubation with the final fraction (LlC1F3). Changes in bioactivity after purification suggest synergistic interactions between quercetin and caffeic acid.
authors.affiliation: Centro de Enseñanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600 Veracruz, Mexico. Electronic address: elkevsf@hotmail.com.
authors.forename: Elke
authors.initials: E
authors.lastname: von Son-de Fernex
authors.affiliation: Centro de Enseñanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600 Veracruz, Mexico. Electronic address: alonsodma@hotmail.com.
authors.forename: Miguel Ángel
authors.initials: MÁ
authors.lastname: Alonso-Díaz
authors.affiliation: Centro Nacional de Investigaciones en Parasitologla Veterinaria, INIFAP, SARH, Km 11.5 Carretera Federal Cuernavaca-Cuautla, Jiutepec, A.P. 206CIVAC, 62500 Morelos, Mexico. Electronic address: pedromdgives@yahoo.com.
authors.forename: Pedro
authors.initials: P
authors.lastname: Mendoza-de Gives
authors.affiliation: Centro de Enseñanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600 Veracruz, Mexico. Electronic address: braulio_36@hotmail.com.
authors.forename: Braulio
authors.initials: B
authors.lastname: Valles-de la Mora
authors.affiliation: Centro De Investigación Biomédica Del Sur, CIBIS, IMSS, Argentina 1, Col. Centro Xochitepec, Morelos, Mexico. Electronic address: gmanases@hotmail.com.
authors.forename: Manases
authors.initials: M
authors.lastname: González-Cortazar
authors.affiliation: Centro De Investigación Biomédica Del Sur, CIBIS, IMSS, Argentina 1, Col. Centro Xochitepec, Morelos, Mexico. Electronic address: azamilpa_2000@yahoo.com.mx.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Zamilpa
authors.affiliation: Centro de Enseñanza Investigación y Extensión en Ganadería Tropical, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Km 5.5 Carretera Federal Tlapacoyan-Martínez de la Torre, C.P. 93600 Veracruz, Mexico. Electronic address: pime11302002@yahoo.com.mx.
authors.forename: Epigmenio
authors.initials: E
authors.lastname: Castillo Gallegos
mesh_terms: D000818:Animals; D000871:Anthelmintics / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D007887:Fabaceae / Q000737:chemistry*; D010063:Ovum / Q000187:drug effects* / Q000648:ultrastructure; D010936:Plant Extracts / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D014251:Trichostrongyloidea / Q000187:drug effects*
chemical_list: D000871:Anthelmintics; D010936:Plant Extracts
keywords: Caffeic acid; Cattle nematodes; Cooperia; Quercetin; SEM; TEM