In vitro anthelmintic activity of the crude hydroalcoholic extract of Piper cubeba fruits and isolated natural products against gastrointestinal nematodes in sheep.

This study describes the in vitro anthelmintic activity of a hydroalcoholic extract from the fruit of Piper cubeba and its major isolated components against the eggs and larvae of gastrointestinal nematodes obtained from naturally-infected ovines. In vitro anthelmintic activity was evaluated using the egg hatch test (EHT), larval development test (LDT) and L3 migration inhibition test (LMT). The extract showed ovicidal and larvicidal activity, with an EC50 of 200 µg/mL and 83.00 µg/mL in the EHT and LDT, respectively. The extract inhibited 100% of larval migration at the lowest tested concentration (95 µg/mL). The crude extract was purified using successive silica gel chromatographic columns, which revealed the lignans hinokinin, cubebin and dihydrocubebin as the major compounds that were present, which were then used in in vitro tests. Cubebin, dihydrocubebin and hinokinin showed higher activity than the crude extract, with an EC50 for ovicidal activity of 150.00 µg/mL, 186.70 µg/mL and 68.38 µg/mL, respectively. In the LDT, cubebin presented an EC50 of 14.89 µg/mL and dihydrocubebin of 30.75 µg/mL. Hinokinin inhibited 100% the larval development at all concentrations evaluated. In the LMT, dihydrocubebin inhibited 100% the larval migration in all concentrations evaluated while cubebin and hinokinin showed EC50 values of 0.89 µg/mL and 0.34 µg/mL, respectively. P. cubeba extract is rich in several classes of active compounds, but here we demonstrate that the described anthelmintic activity may be related to the presence of these lignans, which are present in larger concentrations than other components of the extract. Our results demonstrate for first time the anthelmintic activity against gastrointestinal nematodes in sheep for this class of special metabolites that are present in P. cubeba fruit. However, future detailed studies are needed to evaluate the effectiveness of P. cubeba fruits extract and active lignans in in vivo tests.

In vitro anthelmintic activity of grape pomace extract against gastrointestinal nematodes of naturally infected sheep.

Grape pomace obtained as a byproduct of industrial processing of grapes retains nutrients and substances with anthelmintic potential such as saponins, tannins, and flavonoids. Therefore, this study evaluated the in vitro ovicidal and larvicidal activity of hydroalcoholic grape pomace extract against gastrointestinal nematodes of sheep. The anthelmintic evaluation was performed by in vitro assays with eggs and larvae of nematodes obtained from naturally infected donor sheep. The grape pomace extract showed high in vitro ovicidal and larvicidal activity with LD50 values of 0.30 mg/mL for egg hatching inhibition, 1.01 mg/mL for larval development inhibition and 100% efficacy in larval migration inhibition assays at all concentrations evaluated. The effect of tannins was evaluated by the addition of 50 mg/mL polyvinyl polypyrrolidone to grape pomace extract at the concentration of 12.5 mg/mL. The in vitro ovicidal activity of grape pomace was reduced by only 15% after polyvinyl polypyrrolidone addition, indicating that other phytochemicals also contribute to the ovicidal activity displayed by the extract. Our results demonstrate that grape pomace exhibits in vitro anthelmintic activity, suggesting that, beyond its nutritional potential, this pomace can also be an ally for gastrointestinal nematode control in sheep.