Immune Modulatory Effect of Leonotis Leonurus, Carpobrotus Dimidiatus
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Immune modulatory effect of Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus __________________ Vashka Hurinanthan Submitted in fulfillment of the requirements for the Degree of Master of Technology Biotechnology Department of Biotechnology and Food Technology Faculty of Applied Sciences Durban University of Technology South Africa 2009 Supervisor : Prof. B. Odhav Co-supervisor : Prof. H. Baijnath Date Submitted : February 2009 1 REFERENCE DECLARATION IN RESPECT OF A MASTER’S DISSERTATION I, Vashka Hurinanthan (Student Number: 20415779), Prof. Bharti Odhav and Prof. H. Baijnath do hereby declare that in respect of the following dissertation: Immune modulatory effect of Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus. 1. As far as we can ascertain: (a) No other similar dissertation exists; (b) The only similar dissertation (s) that exists is/are referenced in my dissertation as follows: ________________________________________________________________________ __ ________________________________________________________________________ __ 2. All references as detailed in the dissertation are complete in terms of all personal communications engaged in and public works consulted. V. Hurinanthan Febuary 2009 Student Date B. Odhav Febuary 2009 Supervisor Date H. Baijnath Febuary 2009 Co-Supervisor Dat 2 DEDICATION I dedicate this work to: My late mother, Mrs. P. Hurinanthan, and father Mr. K. P. Hurinanthan, my friends and teachers, for their inspiration and guidance, and to GOD who makes all things possible. 3 ACKNOWLEDGEMENTS I would like to extend my sincere appreciation and gratitude to: My supervisor and mentor, Prof. B. Odhav, Department of Biotechnology and Food Technology, Durban University of Technology, for her guidance and support throughout this project . My co-supervisor, Prof. H. Baijnath, Department of Biology and Conservation, University of Kwa-Zulu Natal, for all his time and effort in collecting my plant material. The Staff of HIV Pathogenesis Programme Laboratories, Doris Duke Medical Centre, University of Kwa Zulu Natal, Nelson Mandela School of Medicine, for all the time and effort in teaching me the immunological techniques. South African Blood Bank (East Coast Region, Pinetown) for the donation of Buffy coats to run these experiments. Ms. R. Gayaram , Mr Sykes and Laboratory assistants at the Insectory Division, Medical Research council, Durban, for their assistance with the anti-mosquito tests. To my friends, Dr Sanker, Dr Thangaraj, Viresh, Alveera, John, Leroosha, Depika and Predashnie, Department of Biotechnology and Food Technology, Durban University of Technology for their support and assistance in the lab throughout this project. The National Research Foundation (NRF, South Africa), for funding this project. My family for their love, support and prayers, during the good and bad times. Nevas Heeralall, my partner, for his encouragement, love and support. 4 Immunomodulatory effect of Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus ABSTRACT Dichrostachys cinerea, Carpobrotus dimidiatus, Capparis tomentosa and Leonotis leonurus are all plants that are indigenous to South Africa. These plants are used in traditional medicine to treat various ailments. However, there is little or no scientific data to justify these traditional uses. Furthermore, it is difficult to reconcile traditional knowledge with scientific evidence because of the overwhelming targeting of signal- responsive systems by plant defensive compounds, multiple sites of action and the connectedness of the signaling pathways, which provide many cures and have pleiotropic effects. In order to evaluate the action spectrum of these plants, and validate its widespread use, this research evaluated the antibacterial, antioxidant, anti-inflammatory, anti-mosquito and immunomodulatory properties of these plants. Antimicrobial activity of the extract was determined by evaluating the bactericidal and fungicidal action using the agar disc diffusion assay. Anti-oxidative properties of the extracts were tested using the DPPH photometric assay. Anti-inflammatory properties were carried out using the 5-lipoxygenase assay. The larvicidal, repellency and insecticidal assay was determined against A.arabiensis. The safe use of these plant extracts was determined by evaluating toxicity, a brine shrimp lethality assay and an in vitro cell culture system using human myelogenous leukemia cell line. Potential carcinogenic activity was evaluated using the Ames Salmonella Mutagenecity assay. The immunomodulatory activity of the extracts on human peripheral blood mononuclear cells 5 was evaluated on freshly harvested lymphocytes using the MTT assay. Cytokine response was evaluated by measuring the secretion of interferon-gamma and interleukin-10. Elucidation of the B cells, T cells, activated T cells, CD 4+, CD 8+ and NK cells was performed by flow cytometry. The extracts showed anti-microbial activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Salmonella typhimurium, Serratia marcescens, Bacillus cereus and Tricoderm sp. The highest activity was shown by methanolic and aqueous extracts of L. leonurus leaves followed by methanolic and aqueous extracts of D. cinerea. Extracts of C. tomentosa and D.cinerea demonstrated a higher degree of free radical scavenging than rutin, which was used as a standard indicating that these plants have strong antioxidant properties. None of the plants showed significant anti-inflammatory activity when compared to NDGA. In the anti-mosquito assays, the extracts showed strong repellency and insecticidal activity. L. leonurus extracts demonstrated the highest insecticidal and repellency activity against the mosquito, and was also found to cause ‗knockdown‘ and mortality. The extracts display no toxicity, cytotoxicity and mutagenicity. The immunological studies for immune modulation showed that the methanol extracts of these plants induce a Th1- predominant immune response because they significantly suppressed the secretion of IL-10 and augment IFN-γ production, which are hallmarks used to indicate a stimulation of the innate immune response. This study also provides new information, with respect to the potential use of these plants in producing a mosquito repellent and an immunostimulant. 6 TABLE OF CONTENTS Page AUTHORS DECLARATION i DEDICATION ii ACKNOWLEDGEMENTS iii ABSTRACT iv TABLE OF CONTENTS vi ABBREVIATIONS x LIST OF TABLES xii LIST OF FIGURES xiv CHAPTER ONE: INTRODUCTION 1.1 Background and aim of the study 1 CHAPTER TWO: LITERATURE REVIEW 2.1 Overview of the plants 2.1.1 Dichrostachys cinerea 3 2.1.2 Carpobrotus dimidiatus 6 2.1.3 Capparis tomentosa 8 2.1.4 Leonotis leonurus 10 2.2 Scientific Rationale 2.2.1 Antibacterial activity 14 2.2.2 Antifungal activity 15 2.2.3 Anti-oxidant activity 16 2.2.4 Anti-inflammatory activity 17 2.2.5 Anti- mosquito activity 18 2.2.6 Safety of traditional medicines 20 2.3 Immunomodulating properties of plants 2.3.1 Immuno-modulation and phytochemicals 23 2.3.2 Stimulation of the immune system 24 2.3.3 Cytokines 26 7 2.3.4 Lymphocytes surface markers 30 2.3.5 CD 4 Cells 30 2.3.6 The role of CD 4 cells in the immune system 31 CHAPTER THREE: METHODOLOGY 3.1 Collection and preparation of plant material 32 3.2 Sample preparation 33 3.3 Biological Screening 3.3.1 Antimicrobial activity 35 3.3.2 Antioxidant activity 37 3.3.3 Anti-inflammatory activity 38 3.3.4 Anti-Mosquito activity 40 3.4 Evaluation of safety of plants 3.4.1 Ames Mutagenicity Test 45 3.4.2 Brine Shrimp Lethality Assay 47 3.4.3 Cytotoxicity 48 3.5 Immuno-modulation 3.5.1 Crude extracts and chemicals 51 3.5.2 Lymphocyte isolation 52 3.5.3 Lymphocyte maintenance 54 8 3.6 Optimization Studies 3.6.1 Optimization of standards and controls 56 3.6.2 Evaluation of the effect of plant extracts on PBMC 57 3.6.3 Lymphocyte proliferation assay 57 3.6.4 Cytokine response of PBMC exposed to plant extracts 58 3.6.5 Effect of plant extracts on T-cell sub-population 59 CHAPTER FOUR: RESULTS 4.1 Biological Activity 4.1.1 Yield and description 61 4.1.2 Antimicrobial activites of the extracts of D. cinerea, C. dimidiatus, C. tomentosa and L. leonurus 65 4.1.3 Antioxidant activity of the extracts of D. cinerea, C. dimidiatus, C. tomentosa and L. leonurus 72 4.1.4 Anti-inflammatory activity of the extracts of D. cinerea, C.dimidiatus, C. tomentosa and L. leonurus 74 4.1.5 Anti-mosquito properties 4.1.5.1 Larvacidal activity 75 4.1.5.2 Repellency activity 77 4.1.5.3 Insecticidal activity 79 4.2 Safety Assays 4.2.1 Ames Mutagenicity Test 82 4.2.2 Brine Shrimp Lethality Assay 86 4.2.3 Cytotoxicity 4.2.3.1 Effect of D. cinerea, C. dimidiatus, C. tomentosa and L. leonurus extracts on K 562 cells 86 4.3 Immmunomodulatory activities of D. cinerea, C. dimidiatus, C. tomentosa and L. leonurus 4.3.1 Optimum concentration of the controls Con A, LPS, PHA, CSA 9 and DMSO 88 4.3.2 Morphological effect of D. cinerea, C. dimidiatus, C. tomentosa and L. leonurus (250 µg/ml) on lymphocytes 4.3.2.1 Methanolic plant extracts 89 4.3.2.2 Aqueous plant extracts 90 4.3.3 Effect of D. cinerea, C. dimidiatus, C.tomentosa and L. leonurus on PBMC 4.3.3.1 Cell viability activity of D.cinerea 91 4.3.3.2 Cell viability activity of C. dimidiatus 92 4.3.3.3 Cell viability activity of C.tomentosa 93 4.3.3.4 Cell viability activity of L. leonurus 94 4.3.3.5 Cell viability activity of L. leonurus flower 95 4.3.4 Immunomodulatory activity evaluated by