In vitro effects of three woody plant and sainfoin extracts on two parasitic stage of 3 parasitic nematode species

Most studies on the effects of tanniferous plants on nematodes have examined forages but have neglected the woody plants. Therefore, in vitro effects of extracts from 3 woody plants (Rubus fructicosus, Quercus robur, Corylus avellana) have been tested on trichostrongyles and compared to sainfoin, a legume forage. Because some in vivo results indicated that the effects of tannins differed depending on the parasitic species and/or stages, the effects were measured on third-stage larvae (L3) and adult worms of Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis. The effects of plant extracts varied according to the plant sources, the parasite species and stages. For the woody plants, significant inhibitory effects were obtained on both stages of abomasal species. Results for T. colubriformis were more variable. Effects of sainfoin extracts were significant on T. colubriformis and H.contortus L3, and on abomasal adult worms. In order to assess the implications of tannins, polyethylene glycol (PEG), an inhibitor of tannins, was added to hazel tree, oak and sainfoin extracts. Without PEG, significant inhibitory effects on L3 and adult worms were confirmed. After addition of PEG, the larval migration and motility of adult worms were restored in most cases. These results confirm variations in effects depending on factors related to plants or parasites and suggest that tannins are partly responsible for the effects

Effect of bioactive compounds from Sainfoin (Onobrychis viciifolia Scop.) on the in vitro larval migration of Haemonchus contortus: role of tannins and flavonol glycosides

Anthelmintic bioactivity against gastrointestinal nematodes has been associated with leguminous forages supporting the hypothesis of a role of condensed tannins. However, the possibility that other compounds might also been involved has received less consideration. Using bio-guided fractionation, the current study aimed at characterising the biochemical nature of the active compounds present in sainfoin (Onobrychis viciifolia), previously identified as an anthelmintic leguminous forage. The effects of sainfoin extracts were evaluated on 3rd stage larvae (L3) of Haemonchus contortus by using a larval migration inhibition (LMI) assay. Comparison of extracts obtained with several solvent systems showed that the bioactivity was associated with the 70:30 acetone/water extract. Further fractionation of the later allowed the separation of phenolic compounds. By use of a dialysis method, compounds were separated with a molecular weight cut-off of 2000 Da. The in vitro anthelmintic effects of the fraction with condensed tannins was confirmed. In the fraction containing molecules of MW < 2000 Da, 3 flavonol glycosides were identified as rutin, nicotiflorin and narcissin. At 1200 μg/ml, each inhibited significantly migration of larvae. Addition of polyvinyl pyrrolidone (PVPP) to both fractions before incubation restore larval migration. These results confirmed the role of both tannins and flavonol glycosides in the anthelmintic properties of sainfoin

Etude in vitro de l’effet des tanins de Newbouldia laevis et de Zanthoxylum zanthoxyloïdes sur la migration des larves infestantes de Haemonchus contortus

Dans le but d&rsquo;aborder le m&eacute;canisme d&rsquo;action des extraits ac&eacute;toniques et &eacute;thanoliques de Newbouldia laevis (Bignoniaceae) et de Zanthoxylum zanthoxylo&iuml;des (Rutaceae), leur effet inhibiteur a &eacute;t&eacute; &eacute;valu&eacute; in vitro sur la migration larvaire de Haemonchus contortus. Le test d&rsquo;inhibition de la migration larvaire (LMI) a &eacute;t&eacute; appliqu&eacute; sur les larves infestantes (L3), &acirc;g&eacute;es de 2 &agrave; 3 mois incub&eacute;es avec des extraits v&eacute;g&eacute;taux &agrave; diff&eacute;rentes concentrations : 150, 300, 600 et 1200 &mu;g/mL mis ou non en contact avec la polyvinylpolypyrrolidone (PVPP). Un t&eacute;moin n&eacute;gatif (tampon PBS) a &eacute;t&eacute; inclus dans chaque test. L&rsquo;observation sous microscope et le d&eacute;nombrement des L3 ayant migr&eacute; par rapport au nombre total de larves d&eacute;pos&eacute;es dans l&rsquo;insert ont permis de calculer le taux de la migration larvaire. Les extraits de Newbouldia laevis et de Zanthoxylum zanthoxylo&iuml;des inhibent in vitro la migration larvaire de Haemonchus contortus. Cet effet est dose-d&eacute;pendant (p&lt;0,001). Les extraits hydro&eacute;thanoliques ont eu plus d&rsquo;effet surtout aux fortes doses. Le contact des extraits des plantes avec la polyvinylpolypyrrolidone (PVPP) annule tout ou une partie de l&rsquo;effet anthelminthique des extraits. Ces r&eacute;sultats sugg&egrave;rent que l&rsquo;inhibition de la migration larvaire est en partie due &agrave; l&rsquo;action des tanins. Le pourcentage d&rsquo;inhibition d&ucirc; aux tanins est de 28,60% quel que soit la plante et quel que soit le solvant d&rsquo;extraction.Keywords: Haemonchus contortus, migration larvaire, tanins, Zanthoxylum zanthoxylo&iuml;des, Newbouldia laevis, B&eacute;ni

Effects of four tanniferous plant extracts on the in vitro exsheathment of third-stage larvae of parasitic nematodes

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previous in vivo and in vitro results suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 of Haemonchus contortus and Trichostrongylus colubriformis. Artificial exsheathment was induced in vitro by adding hypochlorite solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1.5 to 24.7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0.3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 mg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts for T. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts with H. contortus induced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species