Controlling Haemonchus contortus using bioactive compounds from plants with antifungal activity

Abstract

Infection of the gastrointestinal tract of livestock by the nematode parasite Haemonchus contortus is a serious challenge to livestock production. Infected animals, especially sheep and goats, frequently suffer from diarrhoea, anaemia, loss of appetite, reduced body weight and ultimately death if untreated. Anthelmintic drugs are widely used to control nematode infection, but problems of the development of resistance by parasites and unavailability or cost to rural smallholder farmers are serious problems. Their use has also resulted in the presence of residues in meat and milk, which affects food safety. Therefore, alternative control measures such as using plant extracts with putative anthelmintic properties can enhance parasite management. The aim of this study was initially to investigate plant species used in ethnoveterinary medicine to control intestinal parasites for in vitro anthelmintic activity against the sheep nematode Haemonchus contortus. These extracts did not give promising results as the anthelmintic activities were generally low so further work on these plant species was discontinued. The second part of the project was to test plant species with known antifungal activity for activity against H. contortus and a panel of fungal species, as some anthelmintic chemicals also have antifungal activity. For example the benzimidazole group of drugs has demonstrated good correlation between antifungal and anthelmintic activity. Antiparasitic activities of the acetone and water extracts of six plant species with known antifungal activity were determined using the egg hatch assay recommended by the World Association for the Advancement of Veterinary Parasitology (WAAVP). Cytotoxicity was determined by evaluating the viability of cells in the presence of the plant extracts using the tetrazolium-based colorimetric assay against Vero African Green monkey kidney cells. Efficacy of the extracts against various fungi was also tested using a serial microdilution method against three fungal pathogens (Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans), as well as plant fungal pathogens. As parasitic anthelmintic assays are laborious and difficult, a major objective of this study was to investigate if a simple and rapid antifungal assay can be used as a model to isolate anthelmintic compounds from plant extracts. The extracts of Diospyros whyteana and Peddiea africana had the strongest egg hatch inhibitory potential. The extracts had relatively low toxicity to normal mammalian Vero cells. Water extracts were slightly more toxic than acetone crude extracts. All the extracts had good antifungal activities with minimum inhibitory concentration (MIC) values as low as 0.04 mg/ml. Peddiea africana, followed by Schotia brachypetala extracts had the best antifungal activity. Schotia brachypetala inhibited the fungi at 0.04-0.08 mg/ml and the cytotoxicity was low, leading to selectivity indices ranging from 0.59-13.43. The water extract of Cassipourea gummiflua was the most active of all the water extracts. The acetone extracts had a higher number of bioactive compounds than water extracts based on the number of fungal inhibition areas in bioautography. In the anthelmintic egg hatch assay (EHA), the Diospyros whyteana acetone leaf extract had good activity. The extract was also active against Candida albicans with an MIC of 0.04 mg/ml. Candida albicans was therefore used as a model for bioassay-guided fractionation to isolate antifungal compounds from D. whyteana. Successive column chromatography resulted in isolation of three active compounds. Two compounds were not isolated in sufficient quantity to allow structural elucidation and identification but the third compound was identified as a mixture of α-amyrin and β-amyrin. This mixed compound had good antifungal (MIC values as low as 40 μg/ml) as well as anthelmintic activity (30-50% inhibition of egg hatch inhibition of H. contortus). This validates the use of the antifungal assay in bioassay-guided fractionation to isolate anthelmintic compounds in this case. Additional useful activities for anthelmintic or antifungal plant-based remedies include antioxidant activity which may assist the patient in combating the disease. The total phenolic and flavonoid contents of the plant species were investigated, together with antioxidant activity using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay. The total phenolic content of the extracts of Diospyros whyteana (water extract) was the highest followed by Schotia brachypetala (water extract). Generally, water extracts exhibited higher total phenolic contents than the acetone extracts. The plant extracts also generally had relatively high flavonoid contents. Phenolic and flavonoid compounds are often associated with antioxidant activity. The DPPH radical scavenging antioxidant activity of the extracts was less potent than those of the positive controls ascorbic acid and trolox. The acetone extracts of Peddiea africana and Schotia brachypetala had good DPPH radical scavenging ability. This study showed that extracts of some plant species with good antifungal activities also have anthelmintic activity. This may indicate common biological pathways of inhibition or mechanisms of action worthy of further investigation. The use of the antifungal assay in this study assisted with the isolation of anthelmintic compounds. Diospyros whyteana had good anthelmintic and antifungal activity, and compounds active in both assays were isolated and identified from the acetone extract of this species. Further work on other plant species is necessary to confirm the correlation of antifungal and anthelmintic activity in plant extracts.