Discovery of novel plant based compounds to address the drug resistance problem in nematode infested ruminants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Manawatū, New Zealand

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Massey University
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In this thesis, the discovery of novel compounds from plant extracts towards addressing the drug resistance issue in nematode infested ruminants is presented. The nematocidal efficacy of the plant extracts was tested against the L3 stage nematode larvae through bioassay-guided fractionation and chromatographic separation. Quebracho crude powder (QCP) is the only commercially available source of polyphenols. The efficacy of a separated fraction of the QCP was found to be higher (P<0.05) than the crude extract against batches of L3 larvae. However, the research with QCP encountered many challenges and attention was shifted towards medicinal plants found in New Zealand. From a screening study, the Māori plant Piper excelsum (Kawakawa) was found to be the most effective. Of its different components, the leaf component was found to have the highest nematocidal efficacy. Kawakawa (KK) leaf samples were collected over three seasons and the nematocidal efficacy of the leaf samples was found to be independent. From the bioassay-guided fractionation study, the Water and MeOH solvent fractions were found to be most effective, and they were subjected to further reverse-phase chromatographic separation. It was found that the separated fractions had better anthelmintic efficacy than the parent crude solvent fractions (P<0.05). An improvised separation technique named ‘Hand Controlled Countercurrent Separation’ (HCCCS) based on the principle of CCS was developed. It was found that the fractions obtained from the HCCCS study had better anthelmintic efficacy than the parent MeOH-Fraction-4 and the anthelmintic ivermectin (IVM) (P<0.05). A series of combination formulations were made with the separated fractions and IVM. It was observed that these formulations had better efficacy than IVM and the individual HCCCS Fractions (P<0.05). The HCCCS Fractions were subjected to LC-MS/MS investigation and 34 compounds were identified. Of which, 8 were reported in the literature from published KK research. However, the anthelmintic properties of these compounds were not previously reported. The cytotoxicity evaluation of a series of fractions did not reveal any toxic effect to mammalian epithelial cells. Therefore, there is further potential towards isolating leaf fractions into pure isolates which may possess higher nematocidal efficacy compared to the fractions presented in this thesis.
Sheep, Infections, Treatment, Gastrointestinal system, Nematodes, Drug resistance, Medicinal plants, New Zealand