The efficacy of traditionally used Leucosidea sericea (Rosaceae) against Haemonchus contortus and Microbial pathogens

Abstract

Haemonchus contortus is globally the most important economic parasite of sheep and is characterised by its ability to induce severe production losses. Current remedies are faced with resistance and the cost of using these treatments is excessive for poorer/smaller scale farmers. To this extent new anthelmintics that are effective against resistant Haemonchus need to be discovered. From previous studies, it has been established that herbal remedies may serve as a source of these alternate remedies. For this study thirteen tree species use traditionally to treat helminthosis were selected for evaluation. These species were: Brachylaena discolor DC., Apodytes dimidiata E. Mey. ex Arn., Clerodendrum glabrum E. Mey., Clausena anisata (Wild.) Hook.f. ex Benth., Cyathea dregei Kunze, Heteromorpha trifoliata (Spreng.) Cham. & Schltdl. var. abyssinica (A.Rich.), Indigofera frutescens L.f, Leucosidea sericea Eckl. & Zeyh., Milletia grandis E.Mey, Melia azedarach, Maesa lanceolata Forssk., Strychnos mitis S. Moore, Zanthoxylum capense (Thunb). Harv. The antiparasitic activity of leaf extracts of the selected plants species were determined using the egg hatch and larval development inhibitory assay recommended by the World Association for Veterinary Parasitology Guidelines. The selected plant species were also evaluated for possible activity against three fungal pathogens (Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans) and four bacterial pathogens (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa) using a microplate serial dilution method, antioxidant activity using the TEAC method and cytotoxicity was determined using a MTT colorimetric method. From preferential in vitro activity the mechanism of action of the leaf extracts of L. sericea was evaluated through the use of transmission and scanning electron microscopy. In vivo activity of the safe leaf extracts of L.sericea was also concurrently evaluated for efficacy in a sheep model of haemonchosis. Finally in an attempt at isolating active compound(s), the extracts of L. sericea were fractionated into hexane, ethyl acetate, butanol and chloroform and evaluated as for the crude extracts. The best fraction was hereafter selected for isolation of bioactive compounds using open column chromatography. The extracts of three plant species i.e. H. trifoliata, M. lanceolata and L. sericea had EC50 values of 0.62 mg/ml, 0.72 mg/ml and 1.08 mg/ml respectively for the egg hatch assay. Clausena anisata and C. glabrum extracts were active with EC50s of 1.08 mg/ml and 1.48 mg/ml respectively. In the larval development assay the H. trifoliata extract was the most effective with an EC50 of 0.64 mg/ml followed by L. sericea’s 1.27 mg/ml. Based on the cytotoxicity results C. anisata was the least toxic with an LC50 of 0.17 mg/ml, while Cyathea dregei was the most toxic plant with an LC50 of 0.003 mg/ml. The C. anisata extract had the best selectivity index with a value of 0.10 and 0.08 for the two assays, followed by H. trifoliata and L. sericea with values of 0.07, 0.07 and 0.05, 0.04. The C. dregei extract had the worst selectivity index with a value of 0.00019 for both assays. The 13 acetone leaf extracts had good antifungal activities with MIC values as low as 0.02 mg/ml for extracts of C. anisata against A. fumigatus and 0.04 mg/mL for extracts of Z. capense, C. glabrum and M. grandis against A. fumigatus. Clausena anisata extracts had a reasonable selectivity index (2.65) against A. fumigatus. It also had selective activity against A. fumigatus, an overall fungicidal activity of 98% and a total activity of 3395 mL/g against A. fumigatus. Clerodendrum glabrum, Z. capense and M. grandis extracts also had good inhibitory activity of 0.14, 0.09 and 0.28 mg/ml. Maesa lanceolata and L. sericea with an MIC of 0.02 mg/ml had the best antibacterial activity against E. faecalis and P. aeruginosa. Maesa lanceolata had a selectivity index of 5.20, 2.60, 2.60 and 1.30 for P. aeruginosa, E. faecalis, E. coli and S. aureus respectively. Strychnos mitis had a selectivity index of 1.08 for E. coli. Following solvent solvent fractionation the ethyl acetate fractions of L.sericea with EC50 of 0.92 and 0.79 mg/ml against the egg and larvae of H. contortus respectively, cytotoxicity of 283.50 mg/ml and selectivity index of 308.15 and 358.86 for the egg hatch and larval development test respectively was the most active extract. Following isolation using open column chromatography, two phloroglucinol derivatives (agrimol A and G) and β-sitosterol were isolated. The egg hatch and larval development activity for agrimol A was 0.52, 0.08 mg/ml and 0.28, 0.11 mg/l for agrimols G and A. The acetone leaf extracts of L. sericea demonstrated only partial activity in artificially infected H. contortus sheep. Using an extrapolative pharmacokinetic dosage of 109 mg/kg, a 73.1 % reduction in faecal egg count was achieved. At a dosage of 500 mg/kg a reduction of 83.5 % was achieved. In both cases the difference to control group appeared to be non-significant. No clinical signs of toxicity was observed and no mortality from treatment at this dosage. The electron microscopy study identified changes such as the alteration and destruction of the cuticle, changes to the hypodermis, vacuoles within the cytoplasm, cytoplasmic degradation, cellular swelling and in a few cases some abnormality within the mitochondria. This study has confirmed the potential use of a plant L. sericea extract in the treatment of Haemonchus infection in sheep. The study also demonstrated the potential of some anthelmintic extract to be an antifungal. Further studies will be required to evaluate the anthelmintic potential of this extract at higher dosages and against resistant field strains.