An in vitro approach to evaluate the nutraceutical value of plant foliage against Haemonchus contortus.

Nutraceutical plants provide nutrients for the animal as well as secondary compounds that can affect the biology and survival of gastrointestinal nematodes (GIN). Current screening of nutraceutical plants is based on in vitro evidence of anthelmintic (AH) activity against different life stages of GIN, but nutritional information is omitted or scarce. This study proposes an integral in vitro screening protocol to identify the nutraceutical value of the foliage from plant species consumed by small ruminants, using Haemonchus contortus as a biological model. The leaves from Acacia collinsii, A. pennatula, Bunchosia swartziana, Gymnopodium floribundum, Havardia albicans, Leucaena leucocephala, Lysiloma latisiliquum, Mimosa bahamensis, Piscidia piscipula, and Senegalia gaumeri were evaluated for their chemical composition and in vitro digestibility. Acetone:water extracts (70:30) from leaves of each plant were evaluated using the egg hatch assay and larval exsheathment inhibition assay. Respective effective concentrations 50% (EC50) were determined for each assay. The ten plant species showed good nutritional value for ruminants, including crude protein (> 10%), metabolizable energy (> 2.9 MJ/kg DM), and varied CT content (from 1.0 to 37.6%). The best AH activity against H. contortus eggs (EC50 = 401.8 µg/mL) and L3 (EC50 = 83.1 µg/mL) was observed for S. gaumeri extract. Although all the plant species showed in vitro nutraceutical potential, the leaves of S. gaumeri had the best values. The proposed in vitro protocol showed to be useful for the integral assessment of the nutraceutical potential of different plant species as it included the nutritional value and the AH activity against eggs and L3 in the selected plant species.

Is there a negative association between the content of condensed tannins, total phenols, and total tannins of tropical plant extracts and in vitro anthelmintic activity against Haemonchus contortus eggs?

In vitro studies using plant extracts suggest a relationship between their polyphenol contents and their anthelmintic (AH) activity against Haemonchus contortus. High polyphenol content appears to increase the efficacy of plant extracts against H. contortus as assessed by the larval exsheathment inhibition assay (LEIA) while appearing to reduce the AH efficacy measured using the egg hatch assay (EHA). In addition, some plants lack AH activity. Therefore, the present study investigated the relationship between the contents of condensed tannins (CT), total phenols (TP), and total tannins (TT) in methanol:water extracts (70:30) obtained from ten tropical plant species consumed by small ruminants as well as their AH activity against H. contortus evaluated by LEIA and EHA. Extracts of Acacia collinsii, Lysiloma latisiliquum, Havardia albicans, Senegalia gaumeri, Mimosa bahamensis, Piscidia piscipula, Acacia pennatula, Gymnopodium floribundum, Leucaena leucocephala, and Bunchosia swartziana were examined. Positive correlations were found between the effective concentration 50% (EC50) (EHA) of extracts and their CT (r = 0.6809, P < 0.05, n = 10) and TP (r = 0.9152, P < 0.05, n = 10) content, suggesting that their concentration negatively affected AH activity against eggs. Based on the LEIA, there was no significant association between the EC50 and the CT, TP, or TT of all extracts evaluated. Thus, if sheep and goats consume a complex feed mixture with high amounts of CT, TP, and TT, it might be difficult to observe an AH effect against H. contortus egg hatching. However, the AH effect upon L3 establishment might be feasible.

Age of Haemonchus contortus third stage infective larvae is a factor influencing the in vitro assessment of anthelmintic properties of tannin containing plant extracts.

The larval exsheathment inhibition assay (LEIA) of infective larvae (L3) is an in vitro method used to evaluate the anthelmintic (AH) activity of tannin-containing plant extracts against different species of gastrointestinal nematodes, including Haemonchus contortus. Some conditions remain to be defined in order to standardize the LEIA, i.e. the optimal age of larvae produced from donor animals to use in the assays. Therefore, this study aimed at identifying the effect of age and age-related vitality of H. contortus infective larvae produced under tropical conditions, on the in vitro AH activity measured with the LEIA. The same acetone:water (70:30) extract from Acacia pennatula leaves was used to perform respective LEIA tests with H. contortus L3 of different ages (1-7 weeks). Each week, the L3 were tested against different concentrations of extract (1200, 600, 400, 200, 100, 40µg/mL of extract) plus a PBS control. Bioassays were performed with a benzimidazole (Bz) resistant H. contortus (Paraíso) strain. In order to identify changes in L3 vitality on different weeks (1-7), two assays testing larval motility were included only with PBS: the larval migration assay (LMA) and the larval motility observation assay (LMOA). Mean effective concentrations causing 50% and 90% exsheathment inhibition (EC50, EC90) were obtained for every week using respective Probit analyses. On the first week, the larvae had lowest EC50 and EC90 (39.4 and 65.6µg/mL) compared to older larvae (P<0.05). The EC50 and EC90 for weeks 2-5 were similar (P>0.05), while older larvae tended to show higher EC50 and EC90 (P<0.05). Motility showed strong negative correlations with age of larvae (r≥-0.83; P <0.05) and EC50 (r≥-0.80; P<0.05), suggesting that the lower extract efficacy could be associated with decaying vitality of larvae associated with age. More stable efficacy results were found between two to five weeks of age.