Nematocidal Activity of Ethanol and Aqueous Extracts of Persea americana Seeds against Heligmosomoides polygyrus using the Worm Microtracker Method

Abstract

Background. Infections with gastrointestinal helminths constitute a serious obstacle to the good health of the local population in most African Countries. The aim of this study was to evaluate the anthelminthic activity of Persea americana ethanol and aqueous extracts against Heligmosomoides polygyrus using the worm microtracker. Method. Aqueous and ethanolic extracts of P. americana were prepared. Different concentrations of the extracts were tested against the egg and larvae stages of H. polygyrus using an automated high-throughput method. Briefly, embryonated eggs and larvae of this parasite were obtained after the incubation of fresh eggs at 25°C for 24, 48, and 96 hours for embryonated eggs, L1 and L2 larvae, respectively. One hundred microliters of the plant extracts at various concentrations were put in contact in a 96-well microplate with a suspension of 100 embryonated eggs in a total volume of 200 μL and incubated in a worm microtracker where the motility of the worms was recorded every 30 minutes for the ovicidal activity. The final tested extract concentration was 5, 2.5, 1.25, 0.625, and 0.3125 mg/mL, whereas ringer solution (0.95%) and 1.5% Dimethyl sulfoxide (DMSO) were used as negative controls and levamisole as positive control. The same method was used for larvicidal activities. The anthelmintic activity was determined using the average movement of the worms in the tested product compared with the negative control (1.5% DMSO and ringer solution). Results. The egg hatching rates of H. polygyrus had IC50 of 0.49 mg/mL (95% confidence interval: 71.70–92.03) and 0.22 mg/mL (95% confidence interval: 74.28–86.18) for the ethanol and aqueous extract, respectively. These IC50 indicate that the aqueous extract is more active for the inhibition of hatching at a 95% confidence interval. The aqueous and ethanol extracts presented mean inhibitory hatching rates of $78.33\pm 1.67\%$ and $75.67\pm 1.15\%$ at 5 mg/mL, respectively, with no significant differences. The highest percentage of inhibition of L1 larva was observed at 5 mg/mL with $89\pm 2.3\%$ and $85\pm 2.7\%$ for the ethanol and aqueous extracts, respectively. The lowest percentage of inhibition was observed at 0.3125 mg/mL, with $54.67\pm 3.38\%$ and $49\pm 2.64\%$ for the ethanol and aqueous extract, respectively. No significant differences were observed between the two extracts at 5 mg/mL with an inhibitory percentage of $90.67\pm 3.05\%$ (ethanol) and $89.33\pm 2.08\%$ (aqueous). Conclusion. Extracts of P. americana seeds possess nematocidal activity, however, further in silico and in vivo investigations are necessary to confirm their anthelminthic activity.