ANTHELMINTIC, ANTICANCER AND PHYTOCHEMICAL SCREENING OF COTYLEDON ORBICULATA; HERMANNIA DEPRESSA; NICOTIANA GLAUCA AND POTASSIUM PERMANGANATE By Nthatisi Innocentia Molefe Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in the Faculty of Natural and Agricultural Sciences, Department of Zoology and Entomology, University of the Free State Supervisors: Prof. O.M.M. Thekisoe, Dr. A.O.T. Ashafa & Dr. A.M. Tsotetsi December 2013 i SUPERVISORS Prof. Oriel M.M. Thekisoe Parasitology Research Program Department of Zoology and Entomology University of the Free State Qwaqwa Campus Private Bag X13 Phuthaditjhaba 9866 Dr. Anofi O.T. Ashafa Phytomedicine and Phytopharmacology Research Group Department of Plant Sciences University of the Free State Qwaqwa Campus Private Bag X13 Phuthaditjhaba 9866 Dr. Ana M. Tsotetsi Parasites, Vectors and Vector-borne Diseases Programme ARC-Onderstepoort Veterinary Institute Private Bag X05 Onderstepoort 0110 ii DECLARATION I, the undersigned, hereby declare that the work contained in this dissertation is my original work and that I have not previously in its entirety or in part submitted at any university for a degree. I furthermore cede copyright of the dissertation in favour of the University of the Free State. Signature :…………………….. Date :……………………... iii DEDICATION „To the pillar of my strength, my best friend, my everything, my mother, Dieketseng Josephine Molefe for everything she has gone through in order to provide a better future for me and my little brother.‟ iv ACKNOWLEDGEMENTS I thank God, the Almighty who has kept me till this far, who has provided me with strength to pursue life and its short comings until this stage, who has never left nor forsaken me at all times. My deepest gratitude also goes to my supervisors Prof. Oriel Thekisoe; Dr. Tom Ashafa and Dr. Ana Tsotetsi none of this work would have been accomplished if it wasn‟t for them. To my main supervisor, my father and my mentor, Prof. Oriel Thekisoe, I am grateful. He talked sense into me when I did not know where I was heading with life. With all the pressure of work he has put me through I am proud to say he has created a better being in me and I would always be grateful to him for always believing in me and giving hope even when I did not believe in myself, when I was hopeless. He has not only been a supervisor or a mentor but a father to the fatherless like me. “Thank you Prof.” I also thank my family, my mother Dieketseng Molefe, little brother Lehlohonolo Molefe and my grandmother Maria Dladla and lastly my best friend Bangisisi Nyembe. Even though they do not understand what I am doing they have always been by my side with words of encouragements and advices. To fellow students (Ms. Lerato Mabe, Ms. Khethiwe Mtshali, Dr. Petrus Leeto, Mr. Serero Modise, Mr. Moeti Taioe and Mr. Tumisang Mohlakoana) who assisted in making this project a success, they shared jokes that brought back a smile when I did not have any on my face. They became my brothers and sisters who supported and encouraged me to push until I realised my academic objectives. To all of you I say a big thank you. I thank Mrs. Jabu Sithole for administrative support and the motherly care she provided ever since I joined the department. Mr. Manyoni Mabena for technical support during field work and Mr. Emile Bredenhand who has always believed in me and pushed me to the right direction at all times. The research project was funded by the National Research Foundation (NRF) and undertaken at the University of the Free State, Qwaqwa campus. I thank the ARC- Onderstepoort Veterinary Institute for allowing me to use their facilities every now and then when I needed to. Lastly, I thank Ms. Arinna Ferreira and Mr. Thulani Masango from the ARC-OVI and Dr. Nicoline Tanih from the Fort-Hare University for technical training they provided in microbiology. v RESEARCH OUTPUTS Publications 1. Molefe NI, Tsotetsi AM, Ashafa AOT, Thekisoe OMM (2012). In vitro anthelmintic activity of Artemisia afra and Mentha longifolia against parasitic gastro-intestinal nematodes of livestock. Bangladesh Journal of Pharmacology 7:157-163. 2. Molefe NI, Tsotetsi AM, Ashafa AOT, Thekisoe OMM (2013). In vitro anthelmintic activity of Cotyledon orbiculata, Hermannia depressa and Nicotiana glauca extracts against parasitic gastro-intestinal nematodes of livestock. Journal of Medicinal Plants Research 7: 536-542. Conference proceedings 1. Molefe NI, Tsotetsi AM, Ashafa AOT, Thekisoe OMM. In vitro anthelmintic effects of Artemisia afra and Mentha longifolia against parasitic gastro- intestinal nematodes of livestock. The 41st Annual Parasitological Society of Southern Africa (PARSA) Conference, Bloemfontein, South Africa, 1–3 October 2012. 2. Molefe NI, Tsotetsi AM, Ashafa AOT, Thekisoe OMM. In vitro anthelmintic activity of Cotyledon orbiculata, Hermannia depressa and Nicotiana glauca on parasitic gastrointestinal nematodes of livestock. The Annual Indigenous Plant Use Forum (IPUF) Conference, Nelspruit, South Africa, 1- 4 July, 2013 (Second best paper presentation award). vi TABLE OF CONTENTS Title……………………………………………………………………………………….. i Supervisors……………………………………………………………………………… ii Declaration………………………………………………………………………………. iii Dedication……………………………………………………………………………….. iv Acknowledgements…………………………………………………………………….. v Research outputs………………………………………………………………………. vi Table of contents……………………………………………………………………….. vii List of figures……………………………………………………………………………. xii List of plates…………………………………………………………………………….. xiv List of tables…………………………………………………………………………….. xv Abbreviations……………………………………………………………………. …….. xvii Abstract…………………………………………………………………………………... xx Chapter 1. Introduction and literature review 1.1. Medicinal plants and their uses…………………………………………………… 1 1.2. Parasitic gastrointestinal nematodes……………………………………………… 3 1.2.1. Parasitic gastrointestinal nematodes life cycle…………………….…. 5 1.2.2. Economic importance……………………………………………………. 6 1.3. Cancer disease……………………………………………………………………… 7 1.3.1. Development of cancer…………………………………………………... 9 1.3.2. Diagnosis and treatment…………………………………………............ 10 1.4. Choice of plants……………………………………………………………………... 11 1.4.1 Cotyledon orbiculata………………………………………………………. 12 1.4.2 Hermannia depressa……………………………………………………… 13 1.4.3 Nicotiana glauca…………………………………………………………… 14 1.5 Potassium permanganate (KMnO₄)……………………………………………...... 16 1.6. Objectives of the study……………………………………………………………... 17 vii 1.6.1. Statement of the problem………………………………………………… 17 1.6.2. Research hypothesis……………………………………………………... 19 1.6.3. Specific objectives………………………………………………………… 19 Chapter 2. In vitro anthelmintic activity of Cotyledon orbiculata, Hermannia depressa and Nicotiana glauca extracts against parasitic gastrointestinal nematodes of livestock……………………………………………………………..... 20 2.1. Introduction………………………………………………………………………….. 20 2.2. Materials and methods…………………………………………………………….. 21 2.2.1. Plant collection sites…………………………………………………………. 21 2.2.2. Plant material and preparation of extracts………………………………… 21 2.2.3. Faecal sample collection……………………………………………………. 22 2.2.4. Diagnostic methods…………………………………………………………. 23 2.2.5. In vitro assay…………………………………………………………………. 23 2.2.5.1. Egg recovery assay…………………………………………………. 23 2.2.5.2. Egg hatch assay……………………………………………………... 24 2.2.5.3. Larval development assay………………………………………….. 24 2.2.5.4. Larval mortality assay……………………………………………….. 25 2.2.5.5. Larval identification………………………………………………….. 26 2.2.6. Statistical analysis………………………………………………………….... 26 2.3. Results………………………………………………………………………………. 27 2.4. Discussion…………………………………………………………………………… 33 2.5. Conclusions…………………………………………………………………………. 35 Chapter 3. In vitro anticancer activity of medicinal plants of C. orbiculata, H. depressa and N. glauca on breast and cervical cancer cell lines……………... 36 3.1. Introduction………………………………………………………………………….. 36 3.2. Materials and methods……………………………………………………………... 39 3.2.1. Plant collection sites……………………………………………………….. 39 3.2.2. Plant material and preparation of extracts………………………………. 39 3.2.3. Cell lines…………………………………………………………………….. 40 3.2.4. Cell seeding…………………………………………………………………. 40 3.2.5. Cell growth assay…………………………………………………………... 41 3.2.6. Statistical analysis…………………………………………………………. 41 viii 3.3. Results……………………………………………………………………………….. 42 3.4. Discussion…………………………………………………………………………… 46 3.5. Conclusion…………………………………………………………………………... 49 Chapter 4. In vitro and in vivo cytotoxicity analysis of Cotyledon orbiculata, Hermannia depressa and Nicotiana glauca on the bovine kidney cells and brine shrimps……………………………………………………………………………. 50 4.1. Introduction………………………………………………………………………….. 50 4.2. Materials and methods…………………………………………………….............. 52 4.2.1. Plant collection sites………………………………………………………… 52 4.2.2. Preparation of the crude extracts…………………………………………... 52 4.2.3. In vitro cytotoxicity tests……………………………………………………... 53 4.2.3.1. Cell maintenance and preparation……………………………….. 53 4.2.3.2. Cell counts………………………………………………………….. 53 4.2.3.3. MTT assay………………………………………………………….. 53 4.2.3.4. LDH assay………………………………………………………….. 54 4.2.3.5. Data analysis………………………………………………………. 55 4.2.4. In vivo cytotoxicity tests……………………………………………………... 55 4.2.4.1. Hatching of the brine shrimp egg………………………………… 55 4.2.4.2. Data analysis……………………………………………………….. 56 4.3. Results………………………………………………………………………………. 57 4.3.1. In vitro cytotoxicity…………………………………………………………… 57 4.3.2. In vivo cytotoxicity…………………………………………………………… 65 4.4. Discussion…………………………………………………………………………… 70 4.5. Conclusion……………………………………………………………………………