The efficacy of traditionally used Leucosidea sericea (Rosaceae) against Haemonchus contortus and Microbial pathogens Mathew Adamu Thesis submitted in fulfilment of the requirements for the degree Philosophiae Doctor (PhD) In the Phytomedicine Programme Department of Paraclinical Sciences Faculty of Veterinary Science University of Pretoria Promoter: Prof Vinasan Naidoo Co-promoter: Prof JN Eloff December 2012 Declaration I declare that this thesis hereby submitted to the University of Pretoria for the degree Philosophiae Doctor (PhD) has not previously been submitted by me for a degree at this or any other University.That it is my own work in design and in execeution, and that all materials contained herein has been duly acknowledged. Mathew Adamu ii Dedication This thesis is dedicated to the lovely memory of my beloved late Mother, Mrs Margaret Adamu iii Acknowledgements I want to thank Prof JN Eloff who gave me the opportunity to study at the Phytomedicine Programme University of Pretoria. I have learnt tremendously from his wisdom and depth of knowledge that transcend beyond science and research. I also thank him and his wife Christna for opening their doors to me and my family. To both of them we found a foster parent while our stay lasted in South Africa. I also acknowledge the thoroughness of my supervisor Prof V. Naidoo, who exemplified a perfectionist personality to him I am grateful for the thorough and critical review that gave the required depth to this study Thank you for been there for me as a promoter, friend and colleague. My special appreciation to my employers the University of Agriculture Makurdi Nigeria and the Tertiary Education Tax fund for providing me with funding throughout the duration of my studies. The University of Pretoria is acknowledged for awarding me a postgraduate support bursary and funding part of my research work. I thank all my colleagues at the phytomedicine programme and the Faculty of Veterinary Science worthy of mention are; Tharien, Drs Bagla, Aroke, Awouafack, McGaw. I also appreciate Bosvet Pretoria for providing the larvae of Haemonchus contortus which I used for this study. I thank all the staff of the University of Pretoria Biomedical Research Centre especially Jeff and Santa for helping me in the animal study. Erna in the electron microscopy unit and staff of the clinical pathology unit provided valuable assistance. A special thank you to the following colleagues at the University of Agriculture Makurdi Nigeria ; Professors Oyedipe, Onyeyili, Rabo, and Uko, Drs Igoche, Akwuobu, Ishaya, Wachida, and Ofukwu. The following families and friends from Nigeria are appreciated; Ularamu Jubrilla, Dr Adamu, Adamu Isa, Mr and Mrs Dennis Heriju, Mrs Dominic, Aunty Maltina, Mallam Ibrahim, Mr and Mrs Shugaba, Idachaba, Jash, Uncle Patrick. To my Father and all my siblings thank you all for your prayers and support, my Father in-law and his wife for their support and love during this time. I want to specially thank my dear wife Adas and children Vanessa Ampilki, Patrick Ijasini and Margaret Nubwa for their love, prayer, support, care and sacrifices during the entire study period. Lastly but most important I thank God Almighty for the gift of life and good health throughout the duration of my study. iv Publications Mathew Adamu, Vinasan Naidoo, Jacobus N Eloff (2012). Efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa on egg hatching and larval development of Haemonchus contortus. BMC Veterinary Research (In press). Mathew Adamu, Vinasan Naidoo, Jacobus N Eloff (2012). Some southern African plant species used to treat helminth infections in ethnoveterinary medicine have excellent antifungal activities. BMC Complementary and Alternative Medicine 12:213 doi: 10.1186/1472-6882-12-213 Mathew Adamu, Vinasan Naidoo, Jacobus N Eloff. The antibacterial and antioxidant activity of thirteen South African plant species used in ethnoveterinary medicine to treat helminth infections. Prepared for submission to Journal of Medicinal Plant Research. Mathew Adamu, Maurice D. Awouafack, Aroke S. Ahmed, Lyndy J. McGaw, Vinny Naidoo, Jacobus N. Eloff (2012). Anthelmintic phloroglucinol derivatives and antifungal activity of fractions from Leucosidea sericea (Rosaceae). Prepared for submission to Journal of Ethnopharmacology. Mathew Adamu, Jacobus N Eloff, Vinasan Naidoo (2012). Establishing the effects of phloroglucinol derivatives isolated from Leucosidea sericea on the ultrastructure of Haemonchus contortus, using electron microscopy. Prepared for submission to Journal of Experimental Parasitology. Mathew Adamu, Jacobus N Eloff, Vinasan Naidoo (2012). In vivo anthelmintic activity of acetone leaf extract of Leucosidea sericea in sheep artificially infected with Haemonchus contortus. Prepared for submission to Veterinary Parasitology. v Conferences Mathew Adamu, Vinasan Naidoo, Jacobus N Eloff (2012). Efficacy and toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary medicine in South Africa on egg hatching and larval development of Haemonchus contortus. South Africa Association of Botany Conference, University of Pretoria, South Africa 16-18 January, 2012. Oral presentation. Mathew Adamu, Vinasan Naidoo, Jacobus N Eloff (2012). Some southern African plant species used to treat helminth infections in ethnoveterinary medicine have excellent antifungal activities. South Africa Association of Botany Conference, University of Kwazulu-Natal, South Africa 20-24 January, 2013.Oral presentation. Mathew Adamu, Maurice D. Awouafack, Aroke S. Ahmed, Lyndy J. McGaw, Vinny Naidoo, Jacobus N. Eloff (2012). Anthelmintic phloroglucinol derivatives and antifungal activity of fractions from Leucosidea sericea (Rosaceae). South Africa Association of Botany Conference, University of Kwazulu-Natal, South Africa 20-24 January, 2013. Poster Presentation. vi 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. vii 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.