SCREENING OF TRADITIONAL MEDICINAL PLANTS FROM ZIMBABWE FOR PHYTOCHEMISTRY, ANTIOXIDANT, ANTIMICROBIAL, ANTIVIRAL AND TOXICOLOGICAL ACTIVITIES By DENIZ IKLIM VIOL Supervisor: Professor L.S. Chagonda Co-supervisors: Professor R.S. Moyo Professor A.H. Mericli Thesis submitted in partial fulfillment of the requirements for the degree of Master of Philosophy School of Pharmacy College of Health Sciences University of Zimbabwe 2009 ABSTRACT Fourteen indigenous medicinal plants used by traditional medical practitioners in treating sexually transmitted diseases including HIV/AIDS and opportunistic infections were selected after an ethno-botanical pilot survey of five districts from Zimbabwe. The plant materials were collected and extracted separately with methanol. The 28 extracts were lyophilized and screened for phytochemical groups, and biological: antioxidant, antiviral, antibacterial, antifungal and toxicological activities. The phytochemical screening was carried out using Thin Layer Chromatography and UV detection, followed by standard confirmatory tests. The results indicated that seven (25.9%) extracts were positive for alkaloids, ten (35.7%) for anthraquinones, thirteen (46.4%) for coumarins, seventeen (60.7%) for flavonoids, twenty-three (82.1%) for saponins and twenty-five (89.3%) for tannins. Flavonoids, saponins and tannins were the most frequent phytochemical groups found. All extracts contained at least three of the chemical groups. In order to determine the antioxidant activity, the plants were screened for Radical Scavenging Activity using DPPH (2,2-diphenyl-picrylhydrazyl) with β-carotene as reference and their Total Phenolic Contents were measured by the Folin-Ciocalteu reagent using gallic acid as reference. Eight extracts exhibited antioxidant activity with percentages higher than 90% (Rhus chirindensis leaves & roots-both 96.9%; Khaya anthotheca bark-96.1%) and the lowest result was 27.4% for Dichrostachys cinerea roots. Their TPCs ranged from 0.596mg/mg GAE for Khaya anthotheca bark to 0.105mg/mg GAE for Dichrostachys cinerea roots. The phenolic compounds in the extracts correlate with their antiradical activity (r2=0.57), confirming that the phenolics are likely to cause the radical scavenging activity. The antiviral activity was examined using End Point Titration Technique (EPTT) and Neutralisation Test (NT) after calculating the cytotoxicity of the plant extracts on VERO cells. The HSV-2 virus titre was calculated using the Reed and Muench -8.5 method (TCID50 = 10 per 0.1ml). The reduction factor (RF) was calculated and it was considered a promising antiviral result if the RF was ≥ 103. Out of 26 extracts, 13 (50%) showed considerable antiviral activity against the HSV-2 virus. The best results were obtained from the extracts of Dichrostachys cinerea leaves (RF 104), Kigelia africana fruit (RF 104) and Hypoxis rooperi tuber (RF 103) with concentrations ranging from 10.41µg/ml (Dichrostachys cinerea leaves) to 125.0µg/ml (Flacourtia indica roots). The reference acyclovir was active at 1.50µg/ml. Their cytotoxicity could also be beneficiary in developing new anti-tumour drugs. The antibacterial and antifungal activities of the plant extracts (10mg/ml) were investigated by the agar well assay. The chosen microorganisms were Staphylococcus aureus, Streptococcus group A, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus niger. The best results were Terminalia sericea roots, Warburgia salutaris roots, Gymnosporia senegalensis roots and Kigelia africana bark which were active against all micro-organisms. T. sericea roots inhibited the growth of S. aureus with inhibition zone of 7.88±0.48mm where the reference amoxicillin (10μg) gave a zone of 9.00±0.41mm and against P. aeruginosa, gave a larger zone of inhibition, 10.00±0.82mm, than the reference gentamicin (10μg), 7.00±0.40mm. W. salutaris roots were active against both fungal strains with inhibition zones of 10.00±0.82mm for C. albicans and 8.25±0.50mm for A. niger which were even bigger than the zones of the reference amphotericin B (10μg) 6.35±0.50mm and 6.75±0.58mm respectively. The toxicity tests were conducted using the Brine Shrimp (Artemia salina) Lethality Test (BSLT). Five of the extracts showed significant toxicity levels of LC50< 300μg/ml. The lowest readings of LC50, Terminalia sericea leaves (66.7ppm) and Kigelia africana fruit (117.4ppm) were even lower than the positive control, Nerium oleander leaves (141.7ppm) which is a plant with well-established anti-tumour activity. These results confirm the ethno-botanical claims by traditional medical practitioners treating viral, bacterial and fungal infections caused by HIV/AIDS, cancer and cardiovascular diseases with traditional medicinal plants due to the rich phytochemistry, their high levels of antioxidant activity as well as bioactivity of the plants. They should be preserved and harvested with caution not only because of their medicinal value but also the role they play in the rich African heritage. i ACKNOWLEDGEMENTS I would like to give my most sincere gratitude to my supervisor who is also the Director of the School of Pharmacy, Professor L S Chagonda for supporting me throughout the project. I am also grateful to my co-supervisors, Prof R S Moyo in Medical Microbiology at the UZ and Prof A H Mericli, Chairman of Pharmacognosy at the University of Istanbul for their guidance and for availing their laboratory facilities. I want to extend my heartfelt gratitude to all the Chairpersons, the lecturers and the technicians of the following UZ Departments: Pharmacy, Veterinary Sciences, Medical Microbiology, Biochemistry, Food Science and Agriculture, of the Pharmacognosy Department at the University of Istanbul and to the Director of Medicines Control Authority of Zimbabwe. I would also like to thank The National Botanical Gardens for identifying all the plants in this study. Special thanks go to Mrs D. Moyo at the Virology clinic, Faculty of Veterinary Sciences for her tireless support throughout the project. I also would like to acknowledge Dr. F Chinyanganya who helped me to get started in Zimbabwe. I owe a great deal to my workmate Tafadzwa Munodawafa for her astonishing determination and motivation. I thank my lovely sister Dicle Turkoglu who has sent me literature papers all the way from her own university in USA. I am indebted to my dear husband Gordon Viol for his financial and moral support, patience and love. Our sons, Kaan Konrad and Destan Gerhard, are the joys of my life. Most importantly, I dedicate this work to my dear mother and dear father who have done nothing but their best and given their all to make me come this far… This thesis is the result of a project supported largely by the Government of Zimbabwe, Ministry of Environment and Tourism, and by the University of Zimbabwe Research Board. This work was done at the School of Pharmacy, University of Zimbabwe between 2006 and 2008. ii CONTENTS Abstract i Acknowledgments ii List of Tables viii List of Figures ix CHAPTER I 1.0 INTRODUCTION 1 1.1. Traditional Medicine 1 1.2. Drugs of Plant Origin 5 1.3. Phytochemistry 9 1.3.1. Alkaloids 9 1.3.2. Flavonoids 11 1.3.3. Saponins 14 1.3.4. Coumarins 15 1.3.5. Anthraquinones 16 1.3.6. Tannins 17 1.4. Oxidative Stress and Antioxidant Activity 20 1.5. Virology and Antiviral Activity 21 1.5.1. HIV / AIDS 23 1.5.1.1. HIV Life Cycle 24 1.5.1.2. HIV / Antiretroviral Drugs in Clinical Use 26 1.5.1.3. Traditional Medicine Against AIDS 27 1.5.1.4. Herpes Simplex Virus Type-2 28 1.5.1.5. General Information 28 1.5.1.6. Anti-HSV-2 Drugs in Clinical Use 29 iii 1.5.2. Antiviral Susceptibility Testing 30 1.6. Bacteriology, Mycology and Anti-infective Activity 31 1.6.1. Traditional Medicine Against Infections 32 1.6.2. Microorganisms chosen for Study 33 1.6.3. Infections on Body Parts 34 1.7. Toxicology and Bioactivity 35 1.8. Aim of the Study 37 1.9. Objectives of the Study 37 CHAPTER II 2.0 MATERIALS AND METHODOLOGY 39 2.1. Chemicals, Reagents and Equipment 39 2.2. Plant Material 40 2.2.1. Plant Selection Criteria 40 2.2.2. Collection 43 2.3. Plant Extraction 45 2.4. Phytochemical Screening 46 2.4.1. Alkaloids 46 2.4.2. Flavonoids 47 2.4.3. Saponins 49 2.4.4. Coumarins 50 2.4.5. Anthracene Derivatives 50 2.4.6. Tannins 51 2.5. Antioxidant Activity 52 2.5.1. Radical Scavenging Activity 52 2.5.2. Total Phenolic Content Determination 53 iv 2.6. Antiviral Susceptibility Testing 53 2.6.1. Reviving Cell Cultures 54 2.6.2. Subculturing 54 2.6.3. Cell Counting 55 2.6.4. Virus Titration 55 2.6.5. Cytotoxicity 56 2.6.6. Antiviral Screening Assays 56 2.6.6.1. End Point Titration Technique (EPTT) 56 2.6.6.2. Neutralisation Test (NT) 57 2.7. Antimicrobial Susceptibility Testing 58 2.7.1. Sources of microorganisms 58 2.7.2. Antibacterial Screening 58 2.7.3. Antifungal Screening 59 2.7.4. Sensitivity 59 2.8. Toxicity / Bioactivity 60 2.8.1. Hatching the Brine Shrimp 60 2.8.2. Bioassay 60 2.9. Statistical Analysis 61 CHAPTER III 3.0 RESULTS 62 3.1. Plant Extraction 62 3.2. Phytochemical Screening 63 3.3. Antioxidant Activity 66 3.4. Antiviral Screening 70 3.4.1. Cytotoxicity 70 v 3.4.2. Herpes Simplex Virus Type 2 titre 70 3.4.3. Antiviral Assays 70 3.4.3.1. End Point Titration Technique (EPTT) 70 3.4.3.2. Neutralisation Test (NT) 70 3.5. Antimicrobial Susceptibility Testing 72 3.6. Toxicity / Bioactivity Tests 78 3.7. Compilation of Results 79 CHAPTER IV 4.0 DISCUSSION 84 4.1. Phytochemistry Assay 84 4.2.