Antimicrobial interactions of Artemisia afra used in African traditional medicine Sajida Suliman A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the Degree of Master of Pharmacy Johannesburg, 2011 ii Declaration I, Sajida Suliman, declare that this dissertation is my own work. It is being submitted in fulfillment for the degree of Master of Pharmacy at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. ------------------------- Sajida Suliman ------------------------- Date iii Abstract Many therapies prescribed by traditional healers in Southern Africa include plant combinations to treat infectious diseases. Artemisia afra is one of the most commonly used traditional medicines in African traditional medicine and most often given in combination with other plants. This plant?s popularity coupled with its wide range of uses in combination serves as the rationale for the bases of this study. In this study, combinations of A. afra (essential oils and plant extracts), which are commonly used for the treatment of respiratory diseases were studied from an antimicrobial perspective in order to determine if a scientific basis exists for their combined use. The plants used often in double or triple combination with A. afra in the treatment of respiratory tract infections are Lippia javanica, Osmitopsis asteriscoides, Agathosma betulina, Eucalyptus globulus, Allium sativum, Leonotis randii, Tetradenia riparia and Zanthoxylum capense. Essential oils from plant samples were analysed using gas chromatography coupled to mass spectroscopy (GC-MS). Compounds found in highest concentrations were camphor (41.0%) in A. afra, linalool (70.7%) in L. javanica, 1,8-cineole (59.0%) in O. asteriscoides, isomenthone (31.4%) in A. betulina, 1,8-cineole (63.0%) in E. globulus and ?-caryophyllene (32.4%) in T. riparia. Dichloromethane: methanol extracts and aqueous extracts were prepared for each plant using the dried ground plant material collected. The antimicrobial activities of each sample as well as each combination (including essential oils) were tested using the minimum inhibitory concentrations (MIC) assay against a panel of respiratory tract organisms. The highest sensitivities observed for the essential oils were that of E. globulus against Cryptococcus neoformans with a MIC value of 0.6 mg/ml. The dichloromethane: methanol extracts showed the most activity with E. globulus against Moraxella catarrhalis (MIC value of 0.01 mg/ml). The aqueous extracts showed the best activity with Z. capense against Streptococus agalactiae with a MIC value of 0.4 mg/ml. The 1:1 fractional inhibitory concentration (?FIC) values of the combinations of A. afra with L. javanica, A. afra with O. asteriscoides, A. afra with A. betulina, A. afra with E. globulus and A. afra with Z. capense were calculated from the MIC data. Synergy, additivity, iv indifference and antagonistic interactions within the combinations were then interpreted. The most significant interactions of the double combinations with synergistic ?FIC values of 0.2 were the combination of the dichloromethane: methanol extracts of A. afra with O. asteriscoides against Streptococcus pyogenes and the combination of the aqueous extracts of A. afra with E. globulus against Streptococcus pneumoniae. Significant antagonism was noted with the combination of the dichloromethane: methanol extracts of A. afra with E. globulus against Enterococcus faecalis. The ?FIC results of the combinations of A. afra with L. javanica, O. asteriscoides, A. betulina, E. globulus or with Z. capense were used to calculate ratios and plotted on to an isobologram. The isobolograms were interpreted with regard to any synergy, antagonism, or additive interactions present in the combination. Isobolograms revealed the most significant activity with the combination of the aqueous extracts of A. afra with E. globulus against C. neoformans with all the ratios tested being synergistic. The most prominent antagonism (five ratios) noted was in the combination (dichloromethane: methanol extracts) of A. afra with E. globulus against M. catarrhalis. The triple combinations analyzed for their antimicrobial activity were the combinations of A. afra with O. asteriscoides and E. globulus, A. afra with L. randii and E. globulus, A. afra with A. sativum and Z. capense and the combination of A. afra with T. riparia and salt. The most significant synergistic activity was noted for the combination of the essential oils A. afra with T. riparia and salt against Mycobacterium smegmatis with a ?FIC value of 0.2. The combination of A. afra with O. asteriscoides and E. globulus of (dichloromethane: methanol extracts) displayed the most antagonistic activity against M. catarrhalis. When analysing the combinations that include A. afra, it was noted that adjuncts are an important combination ingredient in the traditional method of preparation. These were also tested for their activity. The combinations that include adjuncts i.e. honey, salt, vinegar, brandy and milk showed mainly indifferent interactions. This indifference noted supports the use of these adjuncts by traditional healers as it serves to verify that these adjuncts are at least not hindering the activity of the plant itself, which is a positive direction for future investigations. v Traditional medicine, with regard to A. afra, as prescribed by traditional healers, has commonly employed the use of combinations of more than one plant to treat respiratory conditions. When the antimicrobial activities in combination were examined from a scientific viewpoint, there is evidence of some bases for their traditional use. The results obtained from the testing of the essential oils validate its traditional use as an inhalant. The dichloromethane: methanol extracts showed results varying from synergy to antagonism while the aqueous extracts showed good antimicrobial activity. It is recommended that future studies should be conducted into these interactions to determine the benefits of these combinations for possible use in the commercial and primary health-care sectors. vi Dedication To my parents, Noormohamed and Farida, who have always supported me in my endeavor to achieve success. To my sister, Suraiya, for your unwavering faith and confidence in my abilities. To my nieces Sameeha and Asma, anything is possible. ?The courage of life is often a less dramatic spectacle than the courage of a final moment, but it is no less than a magnificent mixture of triumph and tragedy. People do what they must ? in spite of personal consequences, in spite of obstacles and dangers and pressures ? and that is the basis of all human morality? John F. Kennedy vii Acknowledgements In The Name of Allah (God), the Most Beneficent, the Most Merciful ? Before all, I must first give thanks to my creator and sustainer for guiding me along the path of knowledge and giving me the inner strength and determination to complete this piece of work. This dissertation has shown me that faith is as important a tool in life as skill and determination. ? To my supervisor, Associate Professor Sandy van Vuuren, I thank you from the very bottom of my heart. What I have learnt from you these past few years has been more than simply microbiology and pharmacognosy, but I have learnt the value of hard work, dedication and perseverance. All that I have learnt from you as a Professor as well as a mentor is invaluable. ? To my co supervisor, Professor Alvaro Viljoen, I thank you for your wisdom and boundless knowledge in the field of botany, which proved to be of great value to this research project. My dissertation would not look complete without your photographs so I thank you for providing some of the photographic material of the plants used in my study. ? I would also like to thank all the staff members at the Department of Pharmacy and Pharmacology of the University of the Witwatersrand for all their kind assistance with various tasks. ? Many thanks also go out to the University of the Witwatersrand Health Sciences Library and its willing staff members for their assistance in literature acquisition. ? Thank you to Joanet Maree at the Tshwane University of Technology for all the help with the TLC analysis as well as Dr Guy Kamatou for the assistance with the GC- MS. viii ? The Walter Sisulu Botanical gardens and its staff, in particular Mr Andrew Hankey, were always helpful and hospitable during my visits to collect plant materials. Their understanding and patience is much appreciated. ? Thank you to the National Research Foundation for their generous funding of my research. ? I would also like to thank the University of the Witwatersrand for funding provided. ? To all my friends and colleagues who have been there with me every step on this journey, I thank you for your support and understanding. ? To my sister, Suraiya thank you for your words of wisdom, unwavering support and constant words of encouragement. ? To my brother, Mohamed Ameen, for standing by my side through those long nights and early mornings, thank you. ? I am grateful to my brother Ahmed, and my sister-in law Basheera for giving me encouragement throughout my journey and letting me not waiver in my determination to achieve success. ? To my nieces Sameeha and Asma, you are the coolness of my eyes, thank you for helping me to see the beauty of life and for giving me hope. ? Last but certainly not least, I would like to extend my appreciation and warm gratitude to my parents for their continuous support, financially and for their guidance and wisdom. Mere words are not enough to describe how much I owe to them for everything in my life and I sincerely hope that they understand the gratitude I feel towards them. ix Table of Contents Declaration ii Abstract iii Dedication vi Acknowledgements vii Table of Contents ix List of Figures xvi List of Tables xxii List of Equations xxv Abbreviations xxvi Glossary xxviii CHAPTER 1 30 Introduction 30 1.1 Traditional medicine 30 1.2 Medicinal plants 31 1.3 Combination therapy 32 1.4 Respiratory conditions and plants 35 1.5 Plants used in treating respiratory tract infections 37 1.6 Artemisia afra 40 1.7 A. afra in combination 41 1.8 Aims and objectives of the study 44 CHAPTER 2 46 Materials and methods 46 2.1 Collection of plant material 46 2.2 Sample preparation 47 2.2.1 Dichloromethane: methanol extracts 47 2.2.2 Aqueous extracts 47 2.2.3 Distillation of plant material 47 2.2.4 Adjunct preparation 48 2.3 Chromatographic techniques 49 x 2.3.1 Thin layer chromatography 49 2.3.1.1 Initial TLC Screening 49 2.3.1.2 High performance thin layer chromatography (HPTLC) 50 2.3.1.3 Gas chromatography coupled to mass spectroscopy (GC- MS) 51 2.4 Antimicrobial assays 52 2.4.1 Media and culture preparation 52 2.4.2 Minimum inhibitory concentration (MIC) studies 53 2.4.2.1 Essential oil/extract and antimicrobial control preparation 54 2.4.2.2 Micro-titre plate preparation 55 2.4.3 Combination studies 57 2.4.4 Fractional inhibitory concentration (FIC) determination 57 2.4.5 Isobologram construction 59 CHAPTER 3 62 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Lippia javanica (Burm. F.) Spreng. in combination 62 3.1 Introduction 62 3.2 Results and Discussion 63 3.2.1 Chromatographic techniques 63 3.2.1.1 Essential oil composition of A. afra 63 3.2.1.2 Essential oil composition of L. javanica 64 3.2.1.3 Thin layer chromatography 65 3.2.2 Antimicrobial analysis 68 3.2.2.1 MIC assays and FIC determination 68 3.2.2.1.1 Essential oil 69 3.2.2.1.2 Dichloromethane: methanol extracts 74 3.2.2.1.3 Aqueous extracts 77 3.2.2.2 Isobologram interpretation 80 3.3 Conclusions 84 CHAPTER 4 86 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Osmitopsis 86 xi asteriscoides (L.) Cass. in combination 4.1 Introduction 86 4.2 Results and Discussion 86 4.2.1 Chromatographic techniques 86 4.2.1.1 Essential oil composition of O. asteriscoides 86 4.2.1.2 Thin layer chromatography 87 4.2.2 Antimicrobial analysis 89 4.2.2.1 MIC assays and FIC determination 89 4.2.2.1.1 Essential oils 89 4.2.2.1.2 Dichloromethane: methanol extracts 92 4.2.2.1.3 Aqueous extracts 93 4.2.2.2 Isobologram interpretation 94 4.3 Conclusions 99 CHAPTER 5 101 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Agathosma betulina (P.J. Berguis) Pillans in combination 101 5.1 Introduction 101 5.2 Results and Discussion 101 5.2.1 Chromatographic techniques 101 5.2.1.1 Essential oil composition of A. betulina 101 5.2.1.2 Thin layer chromatography 103 5.2.2 Antimicrobial analysis 104 5.2.2.1 MIC assays and FIC determination 104 5.2.2.1.1 Essential oils 104 5.2.2.1.2 Dichloromethane: methanol extracts 106 5.2.2.1.3 Aqueous extracts 108 5.2.2.2 Isobologram interpretation 109 5.3 Conclusions 114 CHAPTER 6 115 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Eucalyptus globulus Labill. in combination 115 xii 6.1 Introduction 115 6.2 Results and Discussion 115 6.2.1 Chromatographic techniques 115 6.2.1.1 Essential oil composition of E. globulus 115 6.2.1.2 Thin layer chromatography 117 6.2.2 Antimicrobial analysis 119 6.2.2.1 MIC assays and FIC determination 119 6.2.2.1.1 Essential oils 119 6.2.2.1.2 Dichloromethane: methanol extracts 121 6.2.2.1.3 Aqueous extracts 123 6.2.2.2 Isobologram interpretation 124 6.3 Conclusions 129 CHAPTER 7 131 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Zanthoxylum capense (Thunb.) Harv. in combination 131 7.1 Introduction 131 7.2 Results and Discussion 131 7.2.1 Chromatographic techniques 131 7.2.1.1 Thin layer chromatography 131 7.2.2 Antimicrobial analysis 132 7.2.2.1 MIC assays and FIC determination 132 7.2.2.1.1 Dichloromethane: methanol extracts 132 7.2.2.1.2 Aqueous extracts 134 7.2.2.2 Isobologram interpretation 135 7.3 Conclusions 140 CHAPTER 8 141 Artemisia afra in triple plant combinations 141 8.1 Introduction 141 8.2 Results and Discussion 144 8.2.1 A. afra, O. asteriscoides and E. globulus in combination 144 8.2.1.1 Chromatographic techniques 145 xiii 8.2.1.1.1 Thin layer chromatography 145 8.2.1.2 Antimicrobial analysis 146 8.2.1.2.1 MIC assays and FIC determination 146 8.2.1.2.1.1 Essential oils 146 8.2.1.2.1.2 Dichloromethane: methanol extracts 149 8.2.1.2.1.3 Aqueous extracts 151 8.2.2 A. afra, E. globulus and L. randii in combination 153 8.2.2.1 Chromatographic techniques 153 8.2.2.1.1 Thin layer chromatography 153 8.2.2.2 Antimicrobial analysis 155 8.2.2.2.1 MIC assays and FIC determination 155 8.2.2.2.1.1 Dichloromethane: methanol extracts 155 8.2.2.2.1.2 Aqueous extracts 158 8.2.3 A. afra, Z. capense and Allium sativum in combination 160 8.2.3.1 Chromatographic techniques 161 8.2.3.1.1 Thin layer chromatography 161 8.2.3.2 Antimicrobial analysis 162 8.2.3.2.1 MIC assays and FIC determination 162 8.2.3.2.1.1 Dichloromethane: methanol extracts 162 8.2.3.2.1.2 Aqueous extracts 165 8.3 Conclusions 168 CHAPTER 9 170 Artemisia afra in combination with adjuncts 170 9.1 Introduction 170 9.2 Results and Discussion 175 9.2.1 Individual MIC values 175 9.2.2 Honey in combination with A. afra. 175 9.2.3 Skim milk and full cream milk in combination with A. afra. 180 9.2.4 Vinegar and/brandy in combination with A. afra. 182 xiv 9.2.5 Salt in combination with A. afra. 184 9.2.5.1 The combination of A. afra, Tetradenia riparia and salt 186 9.2.5.1.1 Chromatographic techniques 187 9.2.5.1.1.1 Essential oil composition of T. riparia 187 9.2.5.1.1.2 Thin layer chromatography 189 9.2.5.1.2 Antimicrobial analysis 191 9.2.5.1.2.1 Essential oils 191 9.2.5.1.2.2 Dichloromethane: methanol extracts 191 9.2.5.1.2.3 Aqueous extracts 192 9.3 Conclusions 197 CHAPTER 10 198 General conclusion 198 REFERENCES 209 APPENDIX A 248 Monographs of plants studied 248 A1 Agathosma betulina (P.J. Berguis) Pillans 248 A2 Allium sativum L. 251 A3 Artemisia afra Jacq. ex Willd. 253 A4 Eucalyptus globulus Labill. 257 A5 Leonotis. randii S. Moore. 260 A6 Lippia javanica (Burm.f) Spreng 262 A7 Osmitopsis asteriscoides (L.) Cass. 266 A8 Tetradenia riparia (Hochst.) Codd 268 A9 Zanthoxylum capense (Thunb.) Harv. 271 APPENDIX B 274 Conference/publication presentations 274 APPENDIX C 275 Abstracts of presentations/conference presentations 275 xv List of figures Figure 1.1 The relative percentage of research by topic published on A. afra. 40 Figure 1.2 The relative percentages of research by bioactivity published on A. afra. 41 Figure 1.3 A. afra in double combinations. 43 Figure 1.4 A. afra in triple combinations. 44 Figure 2.1 Collection of A. afra in Klipriviersburg. 47 Figure 2.2 Fresh leaf materials being packed into the apparatus. 48 Figure 2.3 Clevenger apparatus being set up. 48 Figure 2.4 Process of HPTLC using the CAMAG Automatic TLC Sampler 4 (ATS4), CAMAG Automatic Development Chamber (ADC2), CAMAG Reprostar (Repro3), CAMAG Chromatogram Immersion Device III and the CAMAG TLC Plate Heater III. 50 Figure 2.5 A typical micro-titre plate. (a) ? negative control; (b) ? positive control. 55 Figure 2.6 Example of the ratios for essential oils and how they were presented in Microsoft Excel ?. 60 Figure 2.7 Example of an isobologram showing synergy ( ), additivity ( ), indifference ( ) and antagonistic ( ) pharmacological interactions. 61 Figure 3.1 TLC of the essential oils of A. afra, L. javanica individually and in combination under UV light of 254 nm, UV light of 365 nm and visualized with vanillin-sulphuric acid reagent. Aa - A. afra; Lj - L. javanica; Aa + Lj -A. afra with L. javanica. 66 Figure 3.2 TLC of the dichloromethane: methanol extracts of A. afra, L. javanica individually and in combination under UV light of 254 nm, under UV light of 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa - A. afra; Lj - L. javanica; Aa + Lj -A. afra with L. javanica. 67 Figure 3.3 TLC of the aqueous extracts of A. afra, L. javanica individually and in combination under UV light of 254 nm, under UV light of 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa - A. afra; Lj - L. javanica; Aa + Lj -A. afra with L. 68 xvi javanica. Figure 3.4 Isobologram of the combination of A. afra with L. javanica essential oils ( ) and the dichloromethane: methanol extracts ( ) against M. catarrhalis (a ? ratio 2:8; b ? ratio 1:9); ? 1:1 combination. 80 Figure 3.5 Isobologram of the combination of A. afra with L. javanica essential oils ( ) and the dichloromethane: methanol extracts ( ) against K. pneumoniae; ? 1:1 combination. 81 Figure 3.6 The MIC values of the different ratios of the combination of A. afra with L. javanica aqueous extracts against M. catarrhalis and K. pneumoniae. 82 Figure 3.7 Isobologram of the combination of A. afra with L. javanica essential oils ( ), dichloromethane: methanol ( ) and aqueous extracts ( ) against E. faecalis (c ? ratio 2:8; d ? ratio 1:9); ? 1:1 combination. 83 Figure 3.8 Isobologram of the combination of A. afra with L. javanica essential oils ( ) dichloromethane: methanol ( ) and aqueous extracts ( ) against C. neoformans (e ? ratio 7:3; f ? ratio 1:9); ? 1:1 combination. 83 Figure 4.1 TLC of the essential oils of A. afra, O. asteriscoides individually and in combination at 254 nm, 365 nm and visualized with vanillin- sulphuric acid reagent. Aa ? A. afra; Oa ? O. asteriscoides; Aa + Oa ? A. afra with O. asteriscoides. 88 Figure 4.2 TLC of the dichloromethane: methanol extracts of A. afra, O. asteriscoides individually and in combination 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Oa ? O. asteriscoides; Aa + Oa ? A. afra with O. asteriscoides. 88 Figure 4.3 TLC of the aqueous extracts of A. afra, O. asteriscoides individually and in combination at 254 nm; 365 nm; visualized under white light; visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Oa ? O. asteriscoides; Aa + Oa ? A. afra with O. asteriscoides. 89 Figure 4.4 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against M. catarrhalis (a ? ratio 1:9; b ? ratio 96 xvii 2:8); ? 1:1combination. Figure 4.5 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against K. pneumoniae (c ? ratio 8:2); ?1:1 combination. 96 Figure 4.6 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against E. faecalis (d ? ratio 9:1); ? 1:1 combination. 97 Figure 4.7 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against C. neoformans (e ? ratio 8:2); ? 1:1 combination. 98 Figure 5.1 TLC fingerprints of the essential oils of A. afra and A. betulina alone and in combination at 254 nm, 365 nm and visualized with vanillin- sulphuric acid reagent. Aa ? A. afra; Ab ? A. betulina; Aa + Ab ? A. afra with A. betulina. 103 Figure 5.2 TLC fingerprints of the dichloromethane: methanol extracts of A. afra and A. betulina alone and in combination at 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Ab ? A. betulina; Aa + Ab ? A. afra with A. betulina. 104 Figure 5.3 Isobologram of the combination of A. afra and A. betulina essential oils ( ) and the dichloromethane: methanol extracts ( ) against M. catarrhalis (a ? ratios 8:2 and 7:3; b ? ratios 3:7, 4:6 and 1:1); ? 1:1 combination. 109 Figure 5.4 Isobologram of the combination of A. afra and A. betulina essential oils ( ) against K. pneumoniae; ? 1:1 combination. 110 Figure 5.5 Isobologram of the combination of A. afra and A. betulina essential oils ( ) against E. faecalis (c ? ratio 9:1); ? 1:1 combination. 110 Figure 5.6 Isobologram of the combination of A. afra and A. betulina essential oils ( ) and dichloromethane: methanol extracts ( ) against C. neoformans (d ? ratio 7:3, e ? ratio 1:9); ? 1:1 combination. 111 xviii Figure 5.7 MIC values of the different ratios of the combination of A. afra with A. betulina dichloromethane: methanol extracts against K. pneumoniae and E. faecalis. 112 Figure 5.8 The MIC values of the different ratios of the combination of A. afra with A. betulina aqueous extracts against K. pneumoniae, M. catarrhalis, E. faecalis and C. neoformans. 113 Figure 6.1 TLC of the essential oils of A. afra and E. globulus individually and in combination at 254 nm, 365 nm and visualized with vanillin- sulphuric acid reagent. Aa ? A. afra; Eg ? E. globulus; Aa + Eg ? A. afra with E. globulus. 117 Figure 6.2 TLC of the dichloromethane: methanol extracts of A. afra and E. globulus individually and in combination at 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Eg ? E.globulus; Aa + Eg ? A. afra with E. globulus. 118 Figure 6.3 TLC of the aqueous extracts of A. afra and E. globulus individually and in combination at 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Eg ? E. globulus; Aa + Eg ? A. afra with E. globulus. 118 Figure 6.4 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against M. catarrhalis; ? 1:1 combination. 125 Figure 6.5 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against K. pneumoniae (a - ratios 4:6 and 5:1); ? 1:1 combination. 126 Figure 6.6 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against E. faecalis (b ? ratios 6:4 and 1:1); ? 1:1 combination. 126 Figure 6.7 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against C. neoformans; ? 1:1 combination. 127 xix Figure 7.1 A. afra dichloromethane: methanol extracts alone and in combination with Z. capense visualizing at 254 nm, 365 nm, white light and after derivatization with vanillin-sulphuric acid. Aa - A. afra; Zc - Z.capense; Aa + Zc - A. afra with Z. capense. 132 Figure 7.2 A. afra aqueous extracts alone and in combination with Z. capense visualizing at 254 nm, 365 nm, white light and after derivatization with vanillin-sulphuric acid. Aa - A. afra; Zc - Z.capense; Aa + Zc - A. afra with Z. capense. 132 Figure 7.3 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) against M. catarrhalis (a ? ratio 2:8 and 1:9); ? 1:1 combination. 136 Figure 7.4 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) against K. pneumoniae; ? 1:1 combination. 137 Figure 7.5 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) against E. faecalis (b ? ratio 6:4); ? 1:1 combination. 137 Figure 7.6 Isobologram of the combination135 of A. afra and Z. capense dichloromethane: methanol extracts139 ( ) and aqueous extracts ( ) against C. neoformans (c ? ratio 7:3); ? 1:1 combination. 138 Figure 7.7 The MIC values of the different ratios of the combination of A. afra with Z. capense aqueous extracts against K. pneumoniae, M. catarrhalis and E. faecalis. 139 Figure 8.1 Summary of the reports available on triple combinations of medicinal plants. 143 Figure 8.2 TLC plates of the dichloromethane: methanol extracts of A. afra, O. asteriscoides and E. globulus alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. 145-146 Figure 8.3 TLC plates of the dichloromethane: methanol extracts of A. afra, L. randii and E. globulus alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. 153-154 Figure 8.4 TLC plates of the dichloromethane: methanol extracts of A. afra, Z. 161-162 xx capense with A. sativum alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. Figure 9.1 The various adjuncts used in traditional medicinal practices. 171 Figure 9.2 The ?FIC interactions of the different types of honey in combination with A. afra. 179 Figure 9.3 A. afra in combination with milk. 181 Figure 9.4 A. afra in combination with vinegar. 183 Figure 9.5 Brandy in combination with A. afra. 184 Figure 9.6 The interaction of A. afra with salt. 186 Figure 9.7 TLC plates of the dichloromethane: methanol extracts of A. afra, Tetradenia riparia with salt alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. 190 Figure A1.1 A. betulina flowers (A.M. Viljoen). 248 Figure A1.2 Botanical distribution of A. betulina (SANBI). 248 Figure A2.1 Bulbules of A. sativum. 251 Figure A3.1 Aerial leaves of A. afra (S.F. van Vuuren) with single feathery leaf of A. afra (inset) (A.M. Viljoen). 253 Figure A3.2 Pale yellowish flowers of A. afra (A.M. Viljoen). 253 Figure A3.3 Geographical distribution of A. afra in South Africa (SANBI). 254 Figure A4.1 Large tree of E. globulus (S.F. van Vuuren). 257 Figure A4.2 Leaves and flowers of E. globulus (S.F. van Vuuren). 257 Figure A5.1 Flower of L. randii (S.F.van Vuuren). 260 Figure A5.2 Distribution map of L. randii (SANBI). 260 Figure A6.1 Leaves of L. javanica with small yellowish white flowers (A.M.Viljoen). 262 Figure A6.2 Geographical distribution of L. javanica in South Africa (SANBI). 262 Figure A7.1 O. asteriscoides leaves and flowers (A.M. Viljoen). 266 Figure A7.2 Geographical distribution of O. asteriscoides (SANBI). 266 Figure A8.1 Glandular leaves of T. riparia (S.F. van Vuuren). 268 Figure A8.2 Flowers of T. riparia (H. de Wet). 268 Figure A8.3 Botanical distribution of T. riparia (SANBI). 269 Figure A9.1 Leaflets of Z. capense (S.F.van Vuuren). 272 Figure A9.2 Geographical distribution of Z. capense in South Africa (SANBI). 272 xxi List of tables Table 1.1 Plant combinations used for the treatment of respiratory infections. 37 Table 2.1 Collection data of the plants studied. 46 Table 2.2 Reagents used and its composition. 49 Table 2.3 Media used in this study to grow bacteria and fungi. 52 Table 2.4 Classification of antimicrobial activity. 57 Table 3.1 GC-MS results of A. afra. 63 Table 3.2 GC-MS results of L. javanica. 65 Table 3.3 MIC values obtained for controls (?g/ml). 68 Table 3.4 MIC and ?FIC values of A. afra, L. javanica essential oils alone and in combination against different pathogens (mg/ml). 69 Table 3.5 MIC and ?FIC values of A. afra and L. javanica dichloromethane: methanol extracts alone and in combination against different pathogens (mg/ml). 75 Table 3.6 MIC and ?FIC values of A. afra, L. javanica aqueous extracts alone and in combination against different pathogens (mg/ml). 78 Table 4.1 GC-MS profile of O. asteriscoides. 87 Table 4.2 MIC (mg/ml) and ?FIC values of A. afra and O. asteriscoides essential oils alone and in combination. 90 Table 4.3 MIC and ?FIC values of A. afra and O. asteriscoides dichloromethane: methanol extracts alone and in combination. 92 Table 4.4 MIC and ?FIC values of A. afra and O. asteriscoides aqueous extracts alone and in combination. 94 Table 5.1 Essential oil composition of A. betulina. 102 Table 5.2 MIC and ?FIC values of essential oils of A. afra and A. betulina, alone and in combination. 105 Table 5.3 MIC and ?FIC values of dichloromethane: methanol extracts of A. afra and A. betulina, alone and in combination. 107 Table 5.4 MIC and ?FIC values of aqueous extracts of A. afra and A. betulina, alone and in combination. 108 Table 6.1 Essential oil constituents of E. globules. 116 Table 6.2 MIC and ?FIC values the essential oils of A. afra and E. globulus alone 120 xxii and in combination. Table 6.3 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra and E. globulus alone and in combination. 122 Table 6.4 MIC and ?FIC values of the aqueous extracts of A. afra and E. globulus alone and in combination. 124 Table 7.1 MIC and ?FIC values of A. afra and Z. capense dichloromethane: methanol extracts independently and in combination. 133 Table 7.2 MIC and ?FIC of A. afra and Z. capense aqueous extracts independently and in combination. 134 Table 8.1 MIC and ?FIC values of the essential oils of A. afra, O. asteriscoides and E. globulus alone and in combination. 148 Table 8.2 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, O. asteriscoides and E. globulus alone and in combination. 150 Table 8.3 MIC and ?FIC values of the aqueous extracts of A. afra, O. asteriscoides and E. globulus alone and in combination. 152 Table 8.4 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, L. randii, and E. globulus alone and in combination. 156 Table 8.5 MIC and ?FIC values of the aqueous extracts of A. afra, L. randii, and E. globulus alone and in combination. 159 Table 8.6 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, Z. capense, and A. sativum alone and in combination. 163 Table 8.7 MIC and ?FIC values of the aqueous extracts of A. afra, Z. capense, and A. sativum alone and in combination. 166 Table 9.1 Various plants used in combination with adjuncts. 172 Table 9.2 Individual mean MIC values of all adjunct samples tested. 175 Table 9.3 MIC values for different types of honey in combination with A. afra samples. 177 Table 9.4 MIC values of A. afra in combination with milk. 180 Table 9.5 A. afra in combination with vinegar and brandy. 182 Table 9.6 MIC values of salt in combination with A. afra. 185 Table 9.7 Essential oil composition of T. riparia. 187 Table 9.8 MIC and ?FIC values of the essential oils A. afra, T. riparia, and salt alone and in combination. 193 xxiii Table 9.9 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, T. riparia, and salt alone and in combination. 194 Table 9.10 MIC and ?FIC values of the aqueous extracts A. afra, T. riparia, and salt alone and in combination. 195 Table 10.1 Summary of the major chemical constituents found from all plant essential oils in this study. 198 Table 10.2 Summary of the most significant antimicrobial activity of the plant samples tested independently. 200 Table 10.3 Summary of the most significant interactions present in the plant combinations tested. 201 Table 10.4 Summary of the adjunct interactions with A. afra. 204 xxiv List of equations Equation 2.1 54 Equation 2.2 58 Equation 2.3 58 Equation 2.4 58 Equation 2.5 59 Equation 2.6 59 xxv Abbreviations AIDS- Acquired Immune Deficiency Syndrome ATCC- American Type Culture Collection CFU- Colony Forming Units CLSI- Clinical and Laboratory Standards Institute DC- Combined volatile and non- volatile constituents that have been prepared with fresh plant material DMSO- Dimethyl Sulfoxide DNA- Deoxyribonucleic acid eV- Electron Volt FIC- Fractional Inhibitory Concentration g- Gram GC- MS- Gas Chromatography Coupled with Mass Spectroscopy HPLC- High Performance Liquid Chromatography HPTLC- High Performance Thin Layer Chromatography hrs-Hours INT- p-Iodonitrotetrazolium Violet min- Minutes MIC- Minimum Inhibitory Concentration mg- Milligram ml- Millilitres m/z- Mass to Charge ratio MRSA- Methicillin Resistant Staphylococcus aureus ND- Not Determined nm- Nano-metres NHLS- National Health Laboratory Services psi- Pounds per Square inch Rf- Distance travelled by spot from baseline with respect to solvent front RRI- Relative Retention Index SAMF- South African Medicines Formulary SANBI- South African National Biodiversity Institute TIC- Total ion Chromatogram xxvi TLC- Thin Layer Chromatography Tr- Trace UV- Ultra Violet WHO- World Health Organisation ?l- Micro-litre ?m- Micro-metre ?C- Degrees Celsius xxvii Glossary Extracts: liquid, powdered or viscous crude mixtures of chemical compounds, extracted from plant material using water or organic solvents e.g. alcohol (ethanol). (van Wyk and Wink, 2004). Thus, the extract contains only the soluble portion of the plant material and the non- soluble (fibrous) residues are discarded. (van Wyk and Wink, 2004). Infusion: (infusum) this refers to a preparation that is made by adding boiling water to the required amount of drug. This preparation is then allowed to steep for five to ten minutes before it is strained. An infusion is sometimes referred to as a ?tea?. (van Wyk and Wink, 2004) Decoction: (decoctum) this refers to a preparation that is made by adding cold water to the required amount of drug. It is then heated to boiling and allowed to simmer for five to ten minutes, thereafter it is strained. (van Wyk and Wink, 2004) Tincture: (tincture) this refers to an alcoholic solution (usually containing 30 to 70% water) prepared from medicinal plant material. This herbal mixture is extracted for a specified period, after which it is pressed and/or strained to separate the liquid and solid material. A mother tincture is often prepared by using 70% ethanol, and the solution is then diluted with clean water to a predetermined yield. (van Wyk and Wink, 2004) Ointments, pastes or gels: these are semisolid preparations intended for external application that contain medicinal substances in a suitable carrier substance (water or oily solvents). (van Wyk and Wink, 2004) Inhalations: they are liquid preparations containing volatile ingredients which when vaporised in a suitable manner, are intended to be brought into contact with the lining of the respiratory tract. The ingredients must be volatile at room temperature, where they might be inhaled from an absorbent pad on which they have been placed, or they may need to be added to water heated to about 65?C, but not boiling water, and the vapour inhaled for 5-10 minutes. (van Wyk and Wink, 2004). xxviii Snuffs: they are preparations of finely powdered, dried medicinal plants that can be drawn up into the nostrils through inhalation. (van Wyk and Wink, 2004). xxix May God have mercy on those who lead the way and those who come behind, and those who fulfil their vows, and those who seek to fulfil them, with His Grace and bounty, His great benefits and favours! For He is the best object of petition and the noblest object of hope; And God is the best protector and the most merciful of those who show mercy, * and the best of friends and the best of heirs and the best replacer of what has been consumed and provider for those devoted who sow and till the soil of good works. And God bless Muhammad and all the Prophets and Messengers! Amen, O Lord of created beings! [Mathn?wi IV, Prologue] in Jewels of Rememberance, Rumi. * Qur?an: Surah Yusuf (Joseph), 12:64. 30 Chapter 1 Introduction 1.1 Traditional medicine Approximately 80% of the world?s population is dependent on traditional medicine (Owalabi et al., 2007; van Wyk et al., 2009). The World Health Organisation (WHO, 2002) has described traditional medicines as ?diverse health practices, approaches, knowledge and beliefs incorporating plant, animal and/or mineral based medicines, spiritual therapies, manual techniques and exercises applied singularly or in combination to maintain well-being, as well as to treat, diagnose or prevent illness? (WHO, 2002). This definition encompasses traditional medicine throughout the world and indicates that the use of medicinal plants is only a small component of a more integrated and holistic approach to health care. However, a general definition does not adequately explain the importance and scope that traditional medicine and traditional healers have on the African continent. From comparisons between Chinese medicines and Ayurvedic treatment, it is evident that many practices have been exchanged between each of these methods of healing (Swerdlow, 2000). These methods have been used for generations and over time have evolved with contributions that can be found in Sri-Lanka, Thailand and Africa (Swerdlow, 2000). These two systems include using one plant for many conditions or many plants to treat just one ailment and remedies are still the mainstay of treatment in the less commercialized and developed areas of India and China, where they continue to show their worth as becoming more popular in the western world (Swerdlow, 2000). The successes that these two ancient medicinal practises have had are not unique as the same principles are applied to traditional healers in Africa. This system has also survived through many generations and has been passed on by teachers, parents and mentors. Traditional healers are widely referred to as medicine men or ?shaman?. These terms are used to denote a person who is skilled in the use of plants and other natural substances and in the rituals of medicine and healing (Swerdlow, 2000). In South Africa, traditional healers are known, most commonly, as ?inyanga? and ?isangoma? (Zulu), ?ixwele? and ?amaquira? (Xhosa), ?nqaka? (Sotho), ?bossiedokter? and ?kruiedokter? (Afrikaans) (Eloff and McGaw in Ahmad et al., 2006). There are an estimated 200 000 indigenous traditional healers in 31 South Africa and approximately 60% of the population consult these healers (van Wyk et al., 2009). This may be due to their large numbers and accessibility as well as the familiarity they have with their patients. As in Western medicine, the variety of different ailments and treatments lends itself to the need for specialization. Thus, under the label of traditional healer, there are herbalists, herb sellers, traditional birth attendants, bone setters, diviners, faith healers, traditional surgeons, spiritualists and many others (Evans, 2002). The fact that traditional healers are an important source of primary health care in Southern Africa is supported by the WHO (2002). The cultural importance and high botanical density in the region are the most likely reasons why the demand is so high (McGaw et al., 2005; Light et al., 2005; Lewu and Afolayan, 2009). Despite the well known fact that traditional medicine is a diverse and often intricate form of health care, its basis is that of holistic healing. All traditional healers treat the psychological as well as physical aspects of each patient. This ensures an all round state of health and wellbeing. The physical symptoms are treated using various medicinal plants alone and in combination. Different parts of these plants may have different effects as they often contain different active ingredients, therefore sometimes the whole plant may be used while at other times only a specific part (van Wyk and Wink, 2004). 1.2 Medicinal plants The traditional medicines that are generally used by the majority of the patients are mostly administered by traditional healers and largely consist of crude plant material and extracts (Reid et al., 2005). In South Africa, as in other developing countries of the world, traditional medicine still forms the backbone of healthcare (Light et al., 2005). The indigenous population, specifically, rely on traditional medicine for all aspects of primary health care (Grierson and Afolayan, 1999; Kelmanson et al., 2000; van Wyk et al., 2009).The use of medicinal plants by the people of South Africa dates back to the early settlement of the native Hottentots. They used many plant species to treat different diseases, (Stone, 1764; Maclagan, 1876; Neuwinger, 2000; Lewu and Afolayan, 2009). Medicinal plants tend to normalize physiological function and correct underlying causes of a disorder instead of temporarily alleviating symptoms (Murray and Pizzorno, 1999; Vermani and Garg, 2002). Generally, medicinal plants are used in two ways. They are sources of biologically important or active compounds (pure, chemically defined active principles) or as 32 an element in complex mixtures containing a broad range of constituents e.g. essential oils, tinctures, or extracts (Hamburger and Hostettman, 1991; Neuwinger, 2000). The modes of administration as well as preparation methods need to be taken into account when studying these medicines. This includes the quantities, the addition of solvents, as well as preparation procedures such as boiling or burning (van Wyk et al., 2009). These vary widely as different parts of the plant are eaten, drunk as teas, or mashed in water for compresses or poultices when freshly harvested. Dried herbs can be soaked in liquids for the same use but are frequently crushed to powder and made into ointments by mixing with fats. These can be massaged into skin scratches or wounds (von Koenen, 2001). 1.3 Combination therapy Even though efficacies between extracts (Kang et al., 1992), essential oils (Lachowicz et al., 1998), and combinations of medicinal plants with conventional antimicrobials (Scott et al., 1995; Shin, 2003; Filoche et al., 2005) have been studied, these have not been done in great depth. Williamson (2001), in a synergy review, has stressed the need for further interactive investigations. This is important in the study of plants such as A. afra as most remedies using such plants are prepared using one or two others together and if studied could prove to have a synergistic effect. Moreover, evidence for synergy or antagonism between three or more plant combinations is sparse (van Vuuren and Viljoen, 2011). Thirty years ago Rahal, (1978) mentioned this fact and until today, not much information has been gathered. When considering all the traditional health systems it becomes apparent that an enormous gap exists between where we are currently looking for new remedies (synthesizing new entities) and where we should be looking i.e. to the tried and tested remedies of old. ?Synergy is what happens when you take two components, combine them and the resulting combination is more potent than the individual components. The result is not just double but many times their singular effect? (Dugmore and van Wyk, 2008). Synergy is defined as the result of the combined action of constituents being greater than expected from a collection of individual contributions (Williamson, 2001). It is important to distinguish between synergy 33 and polyvalent action as the latter is merely the various effects that multiple active constituents produce when used in combination (Williamson, 2001). One of the main reasons for the interest in synergy, by combining two or more plants with antimicrobial activity together, is the need to delay or prevent the increasing emergence of resistant strains of highly pathogenic micro-organisms. The rational for this is that the likelihood of micro-organisms to develop resistance to all the antimicrobials in a combination is low. An example of this in conventional medicine is when treating Tuberculosis; a multiple drug therapy is used to reduce the risk of infection with resistant strains. Another contributing factor is that many antimicrobials produce an unacceptable level of toxicity when used in high doses. Thus, the use of multiple low dose antimicrobial agents is beneficial in reducing the side effects of single high dose compounds (Eliopoulos and Moellering, 1996). Other advantages include the treatment of broad spectrum infections. Among them are Gram- positive, Gram-negative, mycobacterium, yeasts and moulds, all of which can be targeted at the same time. In the ethnobotanical studies of various plants, many publications have noted that synergy is often dependant on the organism tested as well as the doses that are used (Kamatou et al., 2006; van Vuuren et al., 2009; Suliman et al., 2010).Wagner and Ulrich-Merzenich (2009) are of the opinion that whole plant extracts have improved effectiveness as compared to the single constituent isolated from the plant, but due to the complexity of the constituents, it is difficult to identify which component/s are responsible for their synergy (Williamson, 2001). Traditional medical disciplines such as South African traditional healers, Chinese medicine, and Ayurveda, use combinations of different plants to treat various ailments. It is important, therefore, to establish whether the various plants used by traditional healers act in synergy, additively or antagonistically when combined. It is known that medicinal plants have been used since antiquity (Bodeker et al., 1999; Rios and Recio, 2005) to treat common infectious diseases and it has been long acknowledged that essential oils exhibit antimicrobial properties (Finnemore, 1926; Al-Bayati, 2008). Moreover, when using a mixture or combination of plants many more compounds are present and may act at multiple target sites, and therefore may act synergistically to increase effectiveness (Adwan et al., 2006; Al-Bayati, 2008). 34 The major aim of many in vitro testing procedures with traditional medicinal plants is that the focus is on the identification of a single compound. Traditional healers, however, are of the opinion that this is a ?reductionist? approach. They argue that this approach misses the synergism or interactive effects of the multiple ingredients in a complex mixture (Bodeker et al., 1999). Combinations are frequently used in traditional medicine because of the suspected synergism that takes place among the phytochemicals used (Mythilypriya et al., 2008) as well as the aim at curing several diseases simultaneously (Abu-Shanab et al., 2004). Plants may be combined with other components such as animal or insect parts (Reyneke, 1971; Louw et al., 2002), other medicinal plants (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; Felhaber, 1997), and adjuncts such as milk, salt, honey, vinegar, or brandy (Watt and Breyer-Brandwijk, 1962). These combinations are used in order to yield more potent and safer medicines (Reyneke, 1971;; Shin, 2003; Pyun and Shin, 2006). These adjuncts are still used in traditional medicine today, although combinations with other plants have proved to be the most valuable in the treatment of ailments (Louw et al., 2002). The rationale for using plant combinations is the desire to produce a combination that has multiple mechanisms of action, and that the expected action of the combination would be greater than the sum total of the individual or known and unknown chemical components (Harris, 2002). The possibility of other interactions i.e. additivity, indifference or antagonism should also be considered (van Vuuren and Viljoen, 2011). Different plant species in combinations have been scientifically validated. Examples include the commercially marketed combination of Ginkgo biloba with Echinacea wherein synergistic interactions are present (Williamson, 2001). In another study, the combination of Cinnamomum cassia with Allium tuberosum and Cornus officinalis was tested in different ratios. The ratio 8:1:1 (C. officinalis: C. cassia: A. tuberosum) possessed antimicrobial efficacy over a wide range of micro-organisms (Hsieh et al., 2001). Eucalyptus dives and Coriandrum sativum were combined to determine the food preservation efficacy against 12 organisms (Delaquis et al., 2002). Synergy was obtained against Yersinia enterocolitica. Another study combining the essential oils of tea tree with lavender and tested against two dermatophytes. Various ratios of the combination were tested and isobolograms were plotted. The results demonstrated antimycotic activities (Cassella et al., 2002). Kamatou et al. (2006) investigated the combination of the extracts of Salvia chamelaeagnea with Leonotis leonurus 35 to treat respiratory infections. Synergy was observed against the Gram-positive bacteria while antagonism, synergy and/or additive interactions were observed against the Gram-negative bacteria. Rosemary and clove essential oils were combined in the study wherein MIC assays as well as time-kill assays were employed (Fu et al., 2007). Additive interactions were noted using MIC ratios against Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Proteus vulgaris and Pseudomonas aeruginosa. Time-kill studies revealed only concentrations twice that of the MIC values produced a cidal effect. The traditional use of the combination of A. afra with L. javanica essential oil used in traditional African medicine to treat K. pneumoniae related respiratory tract infections was validated by utilising a time-kill assay (van Vuuren, 2007). Al-Bayati, (2008) tested the essential oils and the methanol extracts of the combination of Thymus vulgaris with Pimpinella anisum (Iraqi folk medicine) against nine pathogens. Additive interactions were noted for this combination. Gutierrez et al. (2009) tested the essential oils of Origanum vulgare with Thymus vulgaris and found additivity against food spoilage bacteria. These studies provide some credibility towards the uses of plant combinations for enhanced antimicrobial efficacy. 1.4 Respiratory tract conditions It is commonly noted that traditional healers prescribe the use of plants singularly and in combinations for respiratory infections (Watt and Breyer-Brandewijk, 1962). Therefore, in an effort to improve efficacy of traditional medicines, this study was undertaken in order to establish a scientific basis for the use of the medicinal plants in combination for the treatment of respiratory infections. Respiratory Gram-positive pathogens responsible for infections include Bacillus cereus, Enterococcus faecalis, Mycobacterium smegmatis, Staphylococcus aureus and Streptococcus pyogenes. Gram-negative pathogens associated with respiratory infections include Klebsiella pneumoniae, P. aeruginosa, and Moraxella catarrhalis. Some fungi that cause respiratory infections are Cryptococcus neoformans, Aspergillus niger and Alternaria alternate (van Vuuren, 2007). Details of some the most frequently used test pathogens in this study are given as follows. M. catarrhalis is Gram-negative diplococci and is considered an important cause of upper and lower respiratory tract infections in children and adults. It is reported to be the single cause of sinusitis, otitis media, tracheitis, bronchitis and pneumoniae in children (Verduin et al., 2002). M. catarrhalis is also a common cause of nosocomial infections and causes 36 laryngitis, bronchitis and pneumoniae (Verduin et al., 2002). It is the third most common pathogen after Haemophilus influenza and Streptococcus pneumoniae causing lower respiratory tract infections in adults (Nicotra et al., 1986; Chin et al., 1993). Klebsiella pneumoniae is a Gram-negative encapsulated bacteria, an opportunistic pathogen and causes septicaemia, pneumonia, urinary tract infection, as well as soft tissue infections. It is a prominent nosocomial pathogen causing respiratory tract infections (Podschun and Ullmann, 1998; Brisse et al., 2009). It is one of the leading causes of community-acquired pneumoniae in some countries like South Africa (Ko et al., 2002; Yu et al., 2007; Brisse et al., 2009). E. faecalis is a Gram-positive pathogen and causes up to 90% of enterococcal infections in humans (Kayaoglu and ?rstavik, 2004). It most commonly infects the urinary tract, endocardium causing endocarditis and the abdomen (Boyd and Hoerl, 1981; Jett et al., 1994). It can also infect the lungs, soft tissues, paranasal sinuses and the ears (Rantz and Kirby, 1943; Horvitz and von Graevenitz, 1977; Berk et al., 1983; Maki and Agger, 1988; Doyle and Woodham, 1991; Graninger and Ragette, 1992; Bonten et al., 1993; Jett et al., 1994) Cryptococcus neoformans is an encapsulated yeast, which enables it to reduce size in certain environments, which is thus then ideal for alveolar deposition following inhalation of the pathogen. It is a major pathogen in the immune-compromised patient (causing cryptococcosis) with underlying conditions such as Acquired Immune Deficiency Syndrome (AIDS), lymphoma and patients undergoing corticosteroid therapy (Nielson et al., 1977; Perfect, 1989; Levitz, 1991; Cherniak and Sundstrom, 1994; Diamond, 1995; Hogan et al., 1996; Nosanchuk et al., 2000). It causes asymptomatic pulmonary infections followed by meningitis. When limited to the lungs it causes pneumonia (King and DeWitt, 2009 S. pneumoniae is an alpha haemolytic Gram-positive cocci. Acute respiratory tract infections are often caused by S. pneumoniae, the main aetiological agent. It is exclusive to humans and is one of the most common community-acquired respiratory tract infections requiring antimicrobial therapy. It is also a leading cause of bacterial pneumoniae (File, 2000; Bessen, 2009; WHO, 2009; Farrel et al., 2010). S. pneumoniae causes diseases such as bacteremia, meningitis, pharyngitis, rhinitis, otitis, sinusitis, arthritis and pneumonia (Demachy et al., 2001; Warda et al., 2009). 37 S. pyogenes is referred to as the large colony-forming beta-haemolytic group (Bessen, 2009). It is the causative agent of the ?strep? throat representing pharynhitis and tonsillitis infections. It is also a cause of non-bulbous impetigo in children in underdeveloped countries. Invasive S. pyogenes causes an autoimmune disease Rheumatic fever, with serious cardiac manifestations (Carapetis et al., 2005; Bessen, 2009). Streptococcus agalactiae is a Gram-positive group B streptococcus and is a commensal normal colonizer of the vagina, gastrointestinal and upper respiratory tract of healthy humans. It causes severe disease such as pneumonia, skin and soft tissue infections, septic arthritis and meningitis in immuno-compromised animals and humans (Bessen, 2009; Woods and Levy, 2010). M. smegmatis is a Gram-positive, rapidly growing mycobacterium. It was selected for this study due to its genetic similarity to Mycobacterium tuberculosis and its lack of virulence as an infectious organism (Mitscher and Baker, 1998). M. smegmatis has been associated with soft tissue or wound infections as well as respiratory tract infections (Vonmoos et al., 1986; Newton et al., 1993; Cox et al., 1994; Pennekamp et al., 1997; Ergan et al., 2004). 1.5 Plants used in treating respiratory tract infections Focussing on combinations used for respiratory infections, numerous ethnobotanical remedies are administered. Many of these have not been validated scientifically. A compilation of the ethno-botanical uses of the combinations used for respiratory infections in Southern Africa has been reported and some are summarised in Table 1.1. On examination of these combinations, it is clear that A. afra is predominantly administered in combination. Hence, this study focuses on validating the antimicrobial efficacy of those combinations containing A. afra. Table 1.1 Plant combinations used for the treatment of respiratory infections. Plants in combination Uses Administration Reference A. afra with Eucalyptus globulus Respiratory complaints Crushed leaves or steam from infusions are inhaled or decoctions are taken Watt and Breyer- Brandwijk, (1962); Hutchings et al. (1996). 38 Plants in combination Uses Administration Reference A. afra with Agathosma betulina Respiratory complaints Herbal wine Watt and Breyer- Brandwijk, (1962). A. afra with Zanthoxylum capense The Europeans and Africans use it in febrile conditions, and it is used as a treatment for colds A decoction and an infusion of the leaf is used Watt and Breyer- Brandwijk, (1962). A. afra with Osmitopsis asteriscoides Respiratory complaints Tincture Watt and Breyer- Brandwijk, (1962). A. afra, Eucalyptus globulus with Leonotis microphylla Fever, chest infections and digestive disturbances Infusion Watt and Breyer- Brandwijk, (1962). A. afra, Zanthoxylum capense with Allium sativum Respiratory complaints Decoction Watt and Breyer- Brandwijk, (1962). A. afra with Lippia javanica Fevers, respiratory complaints, measles and as a prophylactic against lung inflammations Infusion, taken with milk Watt and Breyer- Brandwijk, (1962). A. afra, Osmitopsis asteriscoides with Eucalyptus globulus Respiratory complaints Infusion, tincture Watt and Breyer- Brandwijk, (1962); van Wyk et al. (2009). A. afra with Tetradenia riparia and salt Coughs Decoctions Hutchings et al. (1996). A. afra with Alepidea amatymbica Colds and flu Leaves and root/rhizome J?ger, 2003. A. afra with Warburgia salutaris Acute bronchitis, coughs from colds or flu, fever Leaves and bark Felhaber, (1997); J?ger, (2003). A. afra, Alepidea amatymbica with Leonotis leonurus Asthma Leaves, root and leaves Felhaber, (1997). A. afra, Warburgia salutaris with Acorus calamus Chronic bronchitis and emphysema Leaves, bark and rhizome Felhaber, (1997). A. afra, Ruta graveolens with Pteronia divaricata Fever and colds Unknown Hulley et al. (2010). 39 Plants in combination Uses Administration Reference Cheilanthes hirta with Mohria cafforum Colds and sore throats in restless children Burned together and taken in as an inhalation Hutchings et al. (1996). Phoenix reclinata, Euclea natalensis, Capparis tomentosa with Maytennus heterophylla Pleurodynia and pleurisy Steam from the decoction is blown into the wound Hutchings et al. (1996); Bryant, (1966). Cannabis sativa with Warburgia salutaris Dry cough, asthma Powdered bark of Warburgia salutaris and leaves of Cannabis sativa are mixed and smoked Hutchings et al. (1996); Bryant, (1966). Nymphaea nouchali with Tulbaghia species Coughs and colds Decoctions Hutchings et al. (1996). Ranunculus multifidus with Helichrysum nudifolium Severe coughs and sore throats Milk infusions of roots Hutchings et al. (1996). Capparis brassii, Zanthoxylum capense, Capparis tomentosa with what is reported to be Cyrtanthus obliquus but is probably a Eucomis species Chronic coughs Decoction Hutchings et al. (1996); Bryant, (1966). Ozoroa paniculosa with Berchemia zeyheri Acute inflammatory conditions of the chest Taken by mouth Hutchings et al. (1996). Pterocelastrus echinatus with Alepidea amatymbica Respiratory ailments Decoction Hutchings et al. (1996); Pujol, (1990). Cucumis hirsutus with Aster bakeranus Chronic coughs Decoctions Hutchings et al. (1996); Bryant, (1966). Berkheya raphontica with Athrixia phylicoides Dry hacking coughs Decoctions Watt and Breyer- Brandwijk, 1962; Hutchings et al. (1996). Eucalyptus globulus, Corbichonia decumbens with Acorus calamus Blocked nose Boiled infusion Felhaber, (1997). Warburgia salutaris with Acorus calamus Pneumonia Infusion Felhaber, (1997). Lippia javanica with Aloysia triphylla Colds, fever, asthma Herbal teas van Wyk and Wink, (2004). 40 1.6 Artemisia afra (previous studies) A. afra is known to be widely used for medicinal purposes as well as being one of the oldest medicinal plants used in South African traditional medicine (Mangena and Muyima, 1999; Thring and Weitz, 2006). It is considered a future flagship of traditional medicine due to its broad spectrum of activity (Mangena and Muyima, 1999; Liu et al., 2009; van Wyk et al., 2009). A. afra has been documented in the literature for its chemistry, cultivation, toxicology, and quality control. As depicted in Figure 1.1, the bioactivity (53%) of A. afra has been the focus of a majority of studies. Antioxidant (Graven et al., 1992; Burits et al., 2001), antimalarial (Weenen et al., 1990; Kraft et al., 2003; Clarkson et al., 2004; Gathirwa et al., 2007), anti- nematodal (McGaw et al., 2000), cardiovascular (hypotensive) (Guantai and Addae-Mensah, 1999), cytotoxic (Jenett-Siems et al., 2002) and sedative (Nielsen et al., 2004; Stafford et al., 2005) effects have been demonstrated and well documented (van Wyk, 2008a). Figure 1.1 The relative percentage of research by topic published on A. afra. Furthermore, Figure 1.2, which examines the literature pertaining specifically to the biological activity, shows that most of the literature thus far available regarding A. afra?s biological activities have focussed on antimicrobial aspects. 41 Figure 1.2 The relative percentages of research by bioactivity published on A. afra. Some studies conducted on the antimicrobial activity of A. afra include Graven et al. (1992), Gundidza, (1993); Bruneton, (1995); Rabe and van Staden, (1997); Mangena and Muyima, (1999); Huffman et al. (2002); Muyima et al. (2002); J?ger, (2003); Motsei et al. (2003); van Vuuren and Viljoen, (2006); Viljoen et al. (2006b), Vagionas et al. (2007); van Vuuren, (2007). A. afra combinations, however, have been neglected. Further information regarding the botanical description, medicinal uses, geographical locality, chemistry of A. afra and other plants examined in this study may be found in Appendix A3. 1.7 A. afra in combination When examining the research undertaken on A. afra using the Google search engine, ?Artemisia afra? gave 16000 results (7/01/2011). When examining this more closely by using the terminology ?combination?, 1860 results emerged http://www.google.co.za/search?hl=en &biw=1280&bih=683&q=Artemisia+afra&as_q=combination&btnG=Search%C2%A0withi n%C2%A0results). A Pubmed search on ?Artemisia afra? yielded 24 results. Limiting the search to ?Artemisia afra in combination? yielded zero results. Using ScienceDirect, a search on ?Artemisia afra? alone yielded 148 articles and when limited to ?Artemisia afra in combination?, 65 were identified. On Scopus, 70 results were found for ?Artemisia afra? alone and for ?Artemisia afra in combination?, only nine results were obtained. (6/01/2011). 42 This lack of information shows that there is a need for scientifically valid research on this subject. Some instances where plants have been combined with A. afra for the treatment of microbe related infections have been documented in the ethnobotanical literature and are included in Table 1.1. Accounts of the administration of L. javanica with A. afra to treat respiratory disorders have been reported (Hutchings et al., 1996; Watt and Breyer-Brandewijk, 1962). A. afra has also been noted to be used either alone or in combination with the leaves of Eucalyptus spp. by the Xhosa, as an influenza remedy (Watt and Breyer-Brandewijk, 1962). Watt and Breyer-Brandewijk, (1962) sites many examples in which A. afra has been used in combinations with common household herbs e.g. ginger, thyme, rosemary, mint and chamomile. More specialised to traditional healers, as they are widely available and indigenous to the country of South Africa, are combinations involving species such as O. asteriscoides and E. globulus (Watt and Breyer-Brandewijk, 1962). Decoctions of the leaves of A. afra and Agrimonia bracteata are used for colds and decoctions of T. riparia and A. afra with salt are used to treat coughs in Southern Africa (Hutchings et al., 1996; Liu et al., 2009). These combinations sometimes involve two or sometimes three plants. Other examples of combinations with A. afra is the use of A. afra, R. graveolens and P. divaricata, to treat fever and colds (Hulley et al., 2010) other examples include, A. afra leaves and W. salutaris bark for the treatment of cough from cold or flu as well as acute bronchitis (Felhaber, 1997). A combination of A. afra leaves and A. amatymbica root is drunk for colds and flu. Similarly, steam inhalation therapy is used with the combination of A. afra and A. amatymbica rhizome. Asthma is treated with a combination, which is drunk, of A. afra leaves, A. amatymbica root and L. leonurus leaves (Felhaber, 1997). A combination of A. afra leaves, W. salutaris bark, and A. calamus rhizome is used to treat chronic bronchitis and emphysema (Felhaber, 1997). A. afra in combination with other plants in double combination and in triple combinations were analyzed in this study to determine the antimicrobial interactions present. Figure 1.3 shows the double combinations that are tested along with the method of administration. Figure 1.4 shows the triple combinations of A. afra that are evaluated. 43 Artemisia afra Root/leaf infusions b, c Leaf decoctions (with sugar or honey) c Alcohol extract e Steam inhalation c Agathosma betulina Herbal wine a Eucalyptus globulus Steam inhalation Decoctions a and b Osmitopsis asteriscoides Tincture a Infusionh Lippia javanica Infusions with milk or water a and b Zanthoxylum capense Decoction Infusion of leaves a Figure 1.3 A. afra in double combinations. (Watt and Breyer-Brandewijk, 1962a; Hutchings et al., 1996b; van Wyk et al, 2009c; van Wyk and Wink, 2004d; von Koenen, 2001e; Felhaber, 1997f; Moolla, 2006g; Scott and Springfield, 2004h). 44 Figure 1.4 A. afra in triple combinations. (Watt and Breyer-Brandewijk, 1962a; Hutchings et al., 1996b; van Wyk et al, 2009c; van Wyk and Wink, 2004d; von Koenen, 2001e; Felhaber, 1997f; Moolla, 2006g). 1.8 Aims and objectives of the study This study was undertaken with the aim of finding a scientific rationale for the antimicrobial use of A. afra in combination. A further breakdown of objectives for this study is as follows: 1. To identify plants used in combination with A. afra by examination of the ethnobotanical literature. 2. To extract (dichloromethane: methanol and aqueous extracts) and distil collected plant material. 3. To perform gas chromatography coupled with mass spectrometry (GC-MS) on the essential oils obtained from hydrodistillation. 4. To perform thin layer chromatography analysis on the plants essential oils independently and in combination with A. afra. Artemisia afra Root/leaf infusions b,c Leaf decoctions (with sugar or honey) c Alcohol extract e Steam inhalation c Zanthoxylum capense and Allium sativum Decoctions a Eucalyptus globulus and Leonotis randii Infusions a Tetradenia riparia and salt Decoction b Eucalyptus globulus and Osmitopsis asteriscoides Infusions Tincture c 45 5. To carry out antimicrobial screening using minimum inhibitory concentration (MIC) assays on essential oils and extracts. 6. To perform interactive combination studies comparing data obtained from the initial screening antimicrobial studies i.e. with combinations utilizing fractional inhibitory concentration (?FIC) studies and isobologram interpretation. 7. To investigate the use of adjuncts in combination with A. afra using MIC and ?FIC assays. 46 Chapter 2 Materials and Methods 2.1 Collection of plant material Plant species (Table 2.1) were collected (Figure 2.1) at various localities during the summer months between the periods January 2007-April 2007. All plant identities were confirmed by a qualified botanist, either Andrew Hanky or Professor Alvaro Viljoen. The various species and their collection localities are shown in Table 2.1. Voucher specimens are deposited in the Department of Pharmacy and Pharmacology, University of the Witwatersrand. Table 2.1 Collection data of the plants studied. Voucher Species Locality Material distilled (g) Essential oil yield (%w/v) SFVV13 Agathosma betulina Landmeterskop, Middelberg (Mpumalanga) 955 0.9 SFVV14 A. afra Klipriviersburg (Gauteng) 5902.7 0.6 SFVV67 SFVV33 E. globulus Cresta (Gauteng) 3823.4 0.8 SFVV74 SFVV46 L. randii Walter Sisulu Botanical Gardens, Johannesburg (Gauteng) 117.6 No oil SFVV8 L. javanica Fairlands (Gauteng) 2893.3 0.2 SFVV70 SFVV23 O. asteriscoides Hermanus, Cape Town 2233.2 0.8 SFVV47 T. riparia Walter Sisulu Botanical Gardens (Gauteng) 6871.7 0.1 SFVV62 SFVV72 SFVV76 SFVV48 Z. capense Walter Sisulu Botanical Gardens (Gauteng) 262.2 No oil 47 2.2 Sample preparation 2.2.1 Dichloromethane: methanol extracts Extracts were prepared by submerging the dried macerated plant material (?50 g) in a 1:1 mixture of dichloromethane and methanol (CH2Cl2: MeOH) (Merck). This was heated to 37?C for 24 hrs in a water bath. Thereafter, the solution was filtered using cotton wool. The filtered solution was then evaporated by air-drying in a fume hood. The resulting extract was then stored at 4?C for antimicrobial analysis. Figure 2.1 Collection of A. afra in Klipriviersburg. 2.2.2 Aqueous extracts Aqueous extracts were prepared by submerging macerated plant material (?50 g) in sterile distilled water, which was then kept in a water bath at 37?C overnight. Thereafter, it was filtered using Wattman? No 1 filter paper and stored at -80?C for 48 hrs before lyophilisation (van Vuuren and Viljoen, 2006). 2.2.3 Distillation of plant material For the aromatic plants, the essential oils were isolated by conventional hydro-distillation in a Clevenger-type apparatus. A round bottomed flask was packed with fresh leaf material (?1000 g) (Figures 2.2 and 2.3) and ?500 ml of distilled water was added. The flask was then sealed and the cooling system was set up. Thereafter the apparatus was heated using an 48 electric heating mantle. The essential oils were then collected in the condenser and were decanted after three hours. The samples collected were then stored in amber vials and refrigerated for antimicrobial analysis. Figure 2.2 Fresh leaf materials being packed into the apparatus. Figure 2.3 Clevenger apparatus being set up. 2.2.4 Adjunct preparation Salt, powdered skim milk, as well as powdered full cream milk was purchased from the local Checkers? supermarket. The different types of honey used i.e. orange blossom honey, wild blossom honey and blue gum honey were purchased from Woolworths?. The milk powder was made up according to the instructions indicated on the container by the manufacturer. (25.0 mg/ml). The milk was autoclaved and then allowed to cool before analysis. The honey, brandy and vinegar were made up to a concentration of 64.0 mg/ml. The volume of acetone or water to be added was calculated according to Equation 2.2. Honey was also tested at 100% or undiluted. Salt was prepared to a concentration of 4% and diluted with water. 49 2.3 Chromatographic techniques 2.3.1 Thin layer chromatography Thin layer chromatography (TLC) was done as an initial screening process to visualise the chromatographic profile of each plant. Furthermore, combinations of the plants were done in order to qualitatively determine any increase or decrease in compounds when combined. 2.3.1.1 Initial TLC screening A preliminary screening of the plants essential oils, dichloromethane: methanol extracts and the aqueous extracts were performed in order to visualize any compounds present using TLC. In this study for the essential oils, the mobile phase in which the plates were developed comprised of toluene: ethyl acetate (93:7). The mobile phase for development of the dichloromethane: methanol extracts was methanol: water: ethyl acetate (16.5: 13.5: 100) and for the aqueous extracts methanol: water: acetone: ethyl acetate: chloroform (10:8: 30: 40: 12) was the best choice. Compounds resolved on the plate were visualized using either general or specific methods. Ultraviolet light (UV) indicates fluorescent compounds. They are examined at 365 nm (long) and at 254 nm (short) wavelength UV light (Evans, 2002). Alternatively, following development colourless compounds required a chemical reaction in order to visualize their location (Smith and Seakins, 1976). Thus, a spray reagent to produce coloured derivatives was used. For the essential oils, the plates were sprayed with an anisaldehyde-acetic-acid reagent (van Vuuren, 2007) or a vanillin-sulphuric acid reagent (Wagner and Bladt, 1996), as shown in Table 2.2. Similarly, for the extracts, vanillin- sulphuric acid reagent or natural products reagent (Table 2.2) was used (Wagner and Bladt, 1996). This required subsequent heating of the plate to approximately 110?C for 5 min to enable visualization of the compounds (Wagner and Bladt, 1996). Table 2.2 Reagents used and its composition. Reagent Composition Anisadehyde-acetic-acid 0.5 ml anisaldehyde + 10 ml glacial acetic acid + 85 ml methanol Vanillin-sulphuric acid (100 ml ethanol + 1 g vanillin) + (100 ml ethanol + 10 ml sulphuric acid) Natural products- polyethylene glycol 1% methanolic diphenylboric acid + 5% ethanolic poly- ethylene glycol-4000 (Adapted from Wagner and Bladt, 1996). 50 2.3.1.2 High performance thin layer chromatography (HPTLC) After troubleshooting in the initial screening of TLC and optimizing the solvents for the elution of the compounds of the essential oils, dichloromethane: methanol extracts and the aqueous extracts were determined, an automatic TLC system was used. This was performed at the Tshwane University of Technology with the assistance of co-supervisor Professor A.M. Viljoen and Joanet Maree. The plates were then developed in a CAMAG twin trough glass tank pre-saturated with the mobile phase using the CAMAG Automatic Development Chamber (ADC2). The tank was saturated for 10 min in advance and enhanced by keeping filter paper along one wall of the chamber. Each TLC plate was developed to a height of 80 mm (Paramasivam et al., 2009). All TLC runs were carried out under laboratory conditions of approximately 25?C and 39.5% relative humidity. Sample application was performed using the CAMAG Automatic TLC Sampler 4 (ATS4) as shown in Figure 2.4 on pre-activated silica gel HPTLC plates (60GF 254, 20 ? 10 cm). Samples were applied as 10 mm wide bands with N2 flow at 15 ?l/sec, positioned 10 mm from the bottom of the plate and 15 mm from the side of the plate (Paramasivam et al., 2009). Figure 2.4 Process of HPTLC using the CAMAG Automatic TLC Sampler 4 (ATS4)a, CAMAG Automatic Development Chamber (ADC2)b, CAMAG Reprostar (Repro3)c, CAMAG Chromatogram Immersion Device IIId and the CAMAG TLC Plate Heater IIIe. (http://www.hptlc.us/v/products/application/ats4.htmla; http://www.camag.com/v/products/development/adc2.htmlb; a b c d e 51 http://www.camag.com/v/products/assemblies/herbal_kits5.htmlc; http://www.camag.com/v/products/derivatation/derivatization.htmld). After development, plates were dried for five minutes, viewed, and photographed with the CAMAG Reprostar (Repro3). Plates were viewed at 254 nm and 365 nm. Images and full analysis reports are housed in the CAMAG library at Tshwane University of Technology. In order to view the individual compounds, the plates were dipped in vanillin-sulphuric acid reagent (as in Table 2.2) using a CAMAG Chromatogram Immersion Device III and then heated on a CAMAG TLC Plate Heater III at 104?C for 3-5 min. The plates were then photographed using the CAMAG Reprostar (Repro3). 2.3.1.3 Gas chromatography coupled to mass spectroscopy (GC-MS) Essential oil samples were analyzed in order to detect the constituents present. Gas chromatography is considered an efficient method for the characterisation of essential oils (Bakkali et al., 2008; Anwar et al., 2009; Hussain, 2009). The use of gas chromatography coupled with mass spectroscopy (GC-MS) is regarded as a rapid and reliable method in order to identify individual essential oil components (Yadegarinia et al., 2006; Gulluce et al., 2007; Anwar et al., 2009; Hussain, 2009). The oils were analysed by GC?MS (Agilent 6890N GC system coupled directly to a 5973 MS). A volume of 1 ?l was injected using a split ratio (200:1) with an autosampler at 24.79 psi and an inlet temperature of 250?C. The GC system equipped with a HP-Innowax polyethylene glycol column 60 m?250 ?m i.d. ?0.25 ?m film thickness was used. The oven temperature program was 60?C for the first 10 min, rising to 220?C at a rate of 4?C/min and held for 10 min and then rising to 240?C at a rate of 1?C/min. Helium was used as carrier gas at a constant flow of 1.2 ml/min. Spectra were obtained on electron impact at 70 eV, scanning from 35 to 550 m/z. The percentage compositions of the individual components were obtained from electronic integration measurements using flame ionization detection (FID, 250?C). n-Alkanes were used as reference points in the calculation of relative retention indices (RRI). Component identifications were made by comparing mass spectra and retention indices. Library searches were carried out using NIST?, Mass Finder? and Flavour?. 52 2.4 Antimicrobial assays 2.4.1 Media and culture preparation When undertaking microbial susceptibility testing, the cultures used i.e. S. pneumoniae, S pyogenes, S. agalactiae, M. smegmatis, E. faecalis, M. catarrhalis, K. pneumoniae and C. neoformans as well as the media used play an important role in the accuracy of results obtained. The culture medium selected for in vitro susceptibility testing must be able to support good growth of the organism to be tested. In addition, it should not have any effect on the action of the antimicrobial agent being tested (McGinnis and Rinaldi in Lorian, 1991). Table 2.3 lists the media used in the culturing of the bacteria or fungi as well as the strain numbers and characterization of the type of micro-organism. All microbiological techniques, culture and media preparation were followed as in CLSI (2003) guidelines. Table 2.3 Media used in this study to grow bacteria and fungi. The following strains of micro-organisms were obtained from the National Health Laboratory Services (NHLS); E. faecalis ATCC 29212, M. catarrhalis ATCC 23246, K. pneumoniae NCTC 9633, C. neoformans ATCC 90112, S. pneumoniae ATCC 49619, S. pyogenes ATCC 8668, S. agalactiae ATCC 55618 and M. smegmatis (clinical strain). Micro-organism strain number Characterization of type of micro- organism Media S. pneumoniae ATCC 49619 Gram- positive Mueller Hinton (Oxoid) + 2.5% sheep?s blood (National Health Laboratory Services (NHLS) S. pyogenes ATCC 8668 Gram-positive Mueller Hinton (Oxoid) + 2.5% sheep?s blood (NHLS) S. agalactiae ATCC 55618 Gram-positive Mueller Hinton (Oxoid) + 2.5% sheep?s blood (NHLS) M. smegmatis (clinical strain) Gram-positive Middlebrook?7H9-Broth (DifcoTM) + glycerol + Middlebrook ADC Enrichment supplement (BBLTM) E. faecalis ATCC 29212 Gram-positive Tryptone Soya broth/agar (Oxoid) M. catarrhalis ATCC 23246 Gram-negative Tryptone Soya broth/agar (Oxoid) K. pneumoniae NCTC 9633 Gram-negative Tryptone Soya broth/agar (Oxoid) C. neoformans ATCC 90112 Yeast Tryptone Soya broth/agar (Oxoid) 53 2.4.2 Minimum inhibitory concentration (MIC) studies Microbiological assays are performed in order to determine the potency or concentration of a test substance e.g. essential oil or extract to be able to determine its effects on the growth of a micro-organism (Hewitt and Vincent, 2003). A fundamental principle to these assays is that they depend on the comparison of the effect of a standard reference substance e.g. an antibiotic such as ciprofloxacin, and a sample whose potency is to be determined on a ?biological system? e.g. a culture of micro-organisms. The comparison is between the reference standard and the sample of unknown potency i.e. differences in antimicrobial activity can be seen between a known antimicrobial agent and a sample that has never been tested before (Hewitt and Vincent, 2003). One of the simplest and widely preferred techniques used to analyse the effectiveness of an antimicrobial?s action is to determine the minimum inhibitory concentration (MIC) (Eloff, 1998; Hewitt and Vincent, 2003). The minimum inhibitory concentration is the lowest concentration of an antimicrobial that completely inhibits the growth of an organism (Bannister et al., 2000). It is based on the principle of contact of a test organism to a series of dilutions of test antimicrobial/substance (van Vuuren, 2007). Two main methods are used, depending on the substrate used, agar dilution, and the broth dilution method (Hewitt and Vincent, 2003). In the agar, dilution method a serial dilution of the antimicrobial is prepared in molten agar and then plates are poured. The plates are then inoculated uniformly with about 20 ?l of an overnight broth culture containing 105 Colony Forming Units (CFU) of a suitable test organism. After incubation the level with no growth or very few colonies are taken to be the MIC. Controls, e.g. agar plates not containing any antimicrobial as well as a reference antimicrobial with known sensitivity to the micro- organism, must be included in the study (Hewitt and Vincent, 2003). The broth dilution method involves the growth of an organism in a liquid nutrient medium and test solutions at a series of concentrations, from reference standards as well as test substance (Hewitt and Vincent, 2003). A starting concentration of the antimicrobial is made up in broth and then two fold dilutions are serially prepared. Each test tube is then inoculated with the micro-organism of about 105 Colony Forming Units (CFU). After incubation, the tubes are assessed for any microbial growth. The first clear tube after turbidity is recorded as the MIC i.e. the highest dilution of the antimicrobial preventing growth is the MIC of the test 54 organism (Hewitt and Vincent, 2003). This technique requires large quantities of test substance, is very time consuming and when testing extracts, precipitation of the green colour made it difficult to determine the MIC (Eloff, 1998). Thus, a ?sensitive? and ?quick? method specifically developed to obtain MIC values for plant extracts against micro-organisms was developed by Eloff (1998). This microplate or microdilution method is not expensive, approximately 30 times more sensitive than any other method found in literature and it requires small quantities of test substance. It can be used for a large number of samples as well as a variety of different micro-organisms. Controls can be performed on the same plate at the same time and does not require a long time i.e. it is a quick and efficient method (Eloff, 1998). All bacterial cultures were sub-cultured from stock agar plates and grown in the respective broth as shown in Table 2.3 for 24 hrs. The yeast (C. neoformans ATCC 90112) was incubated for 48 hrs. These were prepared using a 1:100 dilution, yielding an approximate inoculum size of 1?106 colony forming units (CFU)/ml). Thus, 500 ?l of test organism was placed into 50 ml of sterile broth. The culture was prepared immediately before use. 2.4.2.1 Essential oil/extract and antimicrobial control preparation Stock solutions of each of the essential oils were made to a starting concentration of 128.0 mg/ml using acetone as a diluent, and extracts were made to a concentration of 64.0 mg/ml. The volume of acetone to be added was calculated using Equation 2.1. Equation 2.1 Positive controls i.e. ciprofloxacin (Sigma-Aldrich) for the bacteria and amphotericin B (Sigma-Aldrich) for the yeasts were also prepared. A starting stock solution of ciprofloxacin was made up with distilled water to a concentration of 0.01 mg/ml. Amphotericin B was diluted with dimethyl sulfoxide (DMSO) initially to a concentration of 1.0 mg/ml. Thereafter distilled water was used to make up a starting stock solution of 0.1 mg/ml. 55 2.4.2.2 Micro-titre plate preparation Under aseptic conditions (laminar flow unit), 100 ?l of sterile water was added to each well of a 96 well micro-titre plate. A negative control (a) i.e. 100 ?l of sterile water was added in row A12 (Figure 2.6) and 100 ?l of conventional antimicrobial (b) (ciprofloxacin for the bacteria and amphotericin B for the yeasts) was placed into well A1 (as shown in Figure 2.5), as positive controls. One hundred micro-litres of each of the prepared test plant samples dissolved in water/acetone were placed in the respective wells in row A. These wells were labeled as depicted in Figure 2.5. Serial dilutions were then done in a vertical orientation, from A to H, using a multi-channel pipette. Thereafter, micro-titre plates were removed from the laminar flow unit and 100 ?l of prepared culture was placed into each if the 96 wells. Micro-titre plates were sealed with sterile adhesive sealing film (AEC Amersham) to prevent evaporation of the essential oils. The remaining prepared culture was incubated along with another negative control, which was a streak plate of the culture used in the assay. This was carried out in order to determine the purity of the culture by the identification of single identical colonies. The micro-titre plates were also incubated with the other samples. Additionally, another negative control, to determine if the media used was sterile whereby the media was left overnight on the desk at 25?C. Incubation was set at 37?C for the bacterial cultures for 24 hrs while the yeast was incubated at 37?C for 48 hrs. Figure 2.5 A typical micro-titre plate. (a) ? negative control; (b) ? positive control. Negative control (a) Se ri a l d il u ti o n s Essential oil or extract Positive control (b) A H 56 Microbial growth or inhibition was detected by using a 0.04% solution of INT (Sigma- Aldrich). An amount of 0.04 g was weighed and added to 100 ml sterile water to make up the required stock solution. INT is insoluble in cold water therefore for optimum solubility. It was placed in a water bath at approximately 55?C for 30 min. Once dissolved, the INT solution was stored at 4?C and used within 2-3 days (Begue and Kline, 1972). After relevant incubation periods, 40 ?l of prepared INT solution was added to each of the 96 wells. For the bacteria, results were recorded six hrs later and for the yeast (C. neoformans) results were recorded 24 hrs later. All tests were performed in duplicate and on separate occasions to ensure accuracy and reproducibility of the results. MIC values were interpreted using the classification adapted from Fabry et al. (1998) Aligiannis et al. (2001) and van Vuuren (2007 and 2008). Fabry et al. (1998) indicated that extracts having an MIC value of ?8.0 mg/ml have some antimicrobial activity. Aligiannis et al. (2001), and Duarte et al. (2005), classified the interpretation of MIC values for plant materials, essential oils and extracts whereby strong inhibitors are those samples that have MIC values up to 0.5 mg/ml. Moderate inhibitors have MIC values between <0.5-1.5 mg/ml and weak inhibitors have MIC values above 1.6 mg/ml. Gibbons (2004) and Rios and Recio, (2005), as described in van Vuuren (2008), have interpreted the MIC value for any natural product below 1.0 mg/ml to have noteworthy antimicrobial activity and an MIC value below 0.1 mg/ml to be ?very interesting?. After extensive reviewing of the literature, van Vuuren (2008) proposed that essential oils with MIC values of 2.0 mg/ml or lower could be considered noteworthy. Thus incorporating all these interpretations, in this study, MIC values are interpreted as follows; an extract having an MIC value below 8.0 mg/ml will be considered to have some antimicrobial activity, but below 1 mg/ml is noteworthy. Any essential oil with an MIC of 2.0 mg/ml or lower will be considered as having noteworthy activity. Furthermore, they can be divided into very strong inhibitors: MIC values less than 0.1 mg/ml; strong inhibitors: MIC values up to 0.5 mg/ml; moderate inhibitors: MIC values between 0.6- 1.5 mg/ml; and weak inhibitors: MIC values above 1.6 mg/ml (Fabry et al., 1998; Aligiannis et al., 2001; Duarte et al., 2005; Rios and Recio, 2005; van Vuuren, 2008). A summary of the classification of antimicrobial activity used in this study is provided in Table 2.4. 57 2.4.3 Combination studies In the testing of combinations of two or more plant essential oils or extracts, in order to assess whether synergy, antagonism or additive interactions are present, three general methods are used (Acar, 2000; Cassella et al., 2002). They are the chequerboard method, time-kill curves and disk diffusion methods (Acar, 2000). Results with killing curves and disk diffusion studies are qualitative assessments of interactions, while the micro-titre-plate method is expressed more quantitatively using the Fractional inhibitory concentration (FIC) method as well as the isobologram method (Acar, 2000). For the purposes of this study, combinations were tested using the micro-titre-plate method as it allows many different combinations and concentrations to be tested (Eliopoulos and Moellering, 1996; Acar, 2000). Table 2.4 Classification of antimicrobial activity. (Adapted from Fabry et al., 1998; Aligiannis et al., 2001; Gibbons, 2004; Duarte et al., 2005; Rios and Recio, 2005; van Vuuren, 2007 and 2008). 2.4.4 Fractional inhibitory concentration (FIC) determination The ?FIC is expressed as the interaction of two plant essential oils or extracts, wherein the concentration of each agent in combination is expressed as a fraction of the concentration that would produce the same effect when used independently (Berenbaum, 1978; van Vuuren and Viljoen, 2008). It is often used to analyze potential antimicrobial interactions. It does however, have certain limitations i.e. the MIC end points are required for each agents being tested (Stergiopoulou et al., 2008). If the MIC end point is not determined at the highest concentration tested, a ?FIC cannot be calculated and thus for the purpose of this study it is Type of sample MIC value (mg/ml) Classification Extracts <8.0 Some antimicrobial activity < 1.0 Noteworthy antimicrobial activity Essential oils ?2.0 Noteworthy antimicrobial activity >2.0 - <8.0 Some antimicrobial activity Essential oils and extracts <0.1 Very interesting antimicrobial activity/ Very strong inhibitors 0.1 - 0.5 Strong inhibitors 0.6 ? 1.5 Moderate inhibitors 58 indicated as ?not determined (1)?. However, results are interpreted by comparing the MIC values obtained for the plant essential oil or extracts alone and in combination. For all combinations where the MIC values were determined, the ?FIC values were calculated according to Equation 2.2 and Equation 2.3 (Berenbaum, 1978; Davidson and Parish, 1989; O?Shaughnessy et al., 2006). Equation 2.2 Equation 2.3 a and b represent the two plants. The sum of the FIC, known as the mean ?FIC index was calculated using Equation 2.4 Equation 2.4 The ?FIC index has been adapted from Berenbaum (1978), Odds (2003) and modified to include an additive interpretation (Schelz et al., 2006; Iten et al., 2009; van Vuuren and Viljoen, 2011). It is used to determine the correlation between the two plants and may be classified as either synergistic (?0.5), additive (0.5 - 1.0), indifferent/non-interactive (>1.0 - 4.0) or antagonistic (?4.0). FIC determination was carried out on all the combinations associated with A. afra. This includes all the double and triple combinations of A. afra with other plants, as well as its combinations with adjuncts such as honey, salt, vinegar, brandy and milk. These combinations were tested and ?FIC values determined against the pathogens used in this study i.e. S. pneumoniae, S. pyogenes, S. agalactiae, M. smegmatis, E. faecalis, M. catarrhalis, K. pneumoniae and C. neoformans, except where an endpoint MIC value was not noted. This was carried out as a preliminary determination of the presence of interactions within a combination for all pathogens. Thereafter four pathogens i.e. K. pneumoniae, M. catarrhalis, E. faecalis and C. neoformans were selected to further investigate interactions present, using isobologram construction studies. Isobologram construction was undertaken, as it may be a more sensitive method to determine the in vitro pharmacodynamic interactions between combinations (Tallarida, 2006; 59 Stergiopoulou et al., 2008). An advantage over FIC determination is that a number of ratios can be tested and plotted out at the same time, in order to detect the interactions at different concentrations of the combination. 2.4.5 Isobologram construction Isobolograms are used to characterize the nature of an interaction between combinations e.g. between plant essential oils or extracts, antibiotics or even between plants and antibiotics (Gennings et al., 1990). An isobol is a level curve, on the surface of an interaction among a combination of plant product extracts or essential oils. The surface is 3-dimensional, wherein the horizontal axes (x and y), represents the individual drug concentrations, and the vertical axes represent the effect of the combination. An isobol is thus the collection of all the points on the surface that lie at a particular effect (Boucher and Tam, 2006). An isobologram is a convenient graphical way of representing the results of a combination study and is useful in visualizing combination data (Tallarida and Raffa, 1995). These different types of interactions have been demonstrated and introduced by Fraser (1870-1871, 1872) and their approach has been extended by Loewe and Muischnek (1926), Loewe (1953) and Berenbaum (1981) (Gennings et al., 1990). More recently, they have been utilized in the combination study between antibiotics and commonly used essential oils by van Vuuren et al. (2009) and by Suliman et al. (2010) to determine the interactions between the essential oil of A. afra with other essential oils commonly used in traditional medicine in South Africa. The isobologram ratio method (Berenbaum, 1978; Williamson, 2001) involves combining the two plants in nine ratios i.e. 9:1; 8:2; 7:3; 6:4; 5:5; 4:6; 3:7; 2:8; 1:9. The MIC was determined for all ratios and the plant samples independently. Micro-titre plates were then prepared as in Section 2.4.2. Ratios were calculated using Equations 2.5 and 2.6 and then plotted onto Microsoft Excel? as is demonstrated in Figure 2.6. Equation 2.5 Equation 2.6 60 MIC values (mg/ml) of plant samples in combination relative to the independent MIC were plotted on an isobologram, using GraphPad Prism? version 5 software, as a ratio, allowing for a graphical representation of the interaction of the various combinations. Figure 2.6 Example of the ratios for essential oils and how they were presented in Microsoft Excel ?. The isobologram can be interpreted by examining the data points of the ratios where the MIC for each concentration is determined in relation to the independent MIC?s (shown as a straight line) and extrapolating synergy (?0.5), additivity (>0.5 ? 1.0), indifference (>1.0 ? ?4.0) and antagonism (>4.0) as shown in Figure 2.7. Tests were undertaken at least in duplicate. Conventional antimicrobials were included in all repetitions as positive controls. Negative controls were also included in the testing as described in Section 2.4.2. Well no Concentrations (mg/ml) MIC values obtained Ratio values Plant X Plant Y Plant X Plant Y Plant X Plant Y 1 32 0 8.00 0.00 1.00 0.00 2 28.8 3.2 7.20 0.80 0.90 0.10 3 25.6 6.4 6.40 1.60 0.80 0.20 4 22.4 9.6 5.60 2.40 0.70 0.30 5 19.2 12.8 4.80 3.20 0.60 0.40 6 16 16 4.00 4.00 0.50 0.50 7 12.8 19.2 3.20 4.80 0.40 0.60 8 9.6 22.4 2.40 5.60 0.30 0.70 9 6.4 25.6 1.60 6.40 0.20 0.80 10 3.2 28.8 0.80 7.20 0.10 0.90 11 0 32 0.00 8.00 0.00 1.00 Ratio values as calculated 100% test substance (plant X) Well no Concentrations (mg/ml) MIC values obtained Ratio values Plant X Plant Y Plant X Plant Y Plant X Plant Y 1 32 0 8.00 0.00 1.00 0.00 2 28.8 3.2 7.20 0.80 0.90 0.10 3 25.6 6.4 6.40 1.60 0.80 0.20 4 22.4 9.6 5.60 2.40 0.70 0.30 5 19.2 12.8 4.80 3.20 0.60 0.40 6 16 16 4.00 4.00 0.50 0.50 7 12.8 19.2 3.20 4.80 0.40 0.60 8 9.6 22.4 2.40 5.60 0.30 0.70 9 6.4 25.6 1.60 6.40 0.20 0.80 10 3.2 28.8 0.80 7.20 0.10 0.90 11 0 32 0.00 8.00 0.00 1.00 100% test substance (plant X) 100% test substance (plant Y) 100% test substance (plant Y) Ratios ranging from 9:1 to 1:9 Ratios ranging from 9:1 to 1:9 61 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 MIC of Plant X in combination/ MIC of Plant X independently M IC o f P la nt Y in c om bi na tio n/ M IC o f P la nt Y in de pe nd en tly Figure 2.7 Example of an isobologram showing synergy ( ), additivity ( ), indifference ( ) and antagonistic ( ) pharmacological interactions. 62 Chapter 3 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Lippia javanica (Burm. f.) Spreng. in combination 3.1 Introduction A. afra is combined with many different plants in South African traditional medicine, but most notably and consistently of all, with Lippia javanica. (Watt and Breyer-Brandwijk, 1962; Neuwinger, 2000; Huffman et al., 2002). A decoction of L. javanica is generally mixed with a decoction of wormwood and used for colds and influenza (Smith, 1895). Likewise, an infusion made with A. afra and L. javanica, is given to treat fevers, influenza (Hutchings et al., 1996), measles and used as a prophylactic against lung inflammations (Smith, 1895; Watt and Breyer-Brandwijk, 1962). Although there has been much citation in literature regarding the use of the combination of these two plants, there is very little scientific evidence to support the traditional use in combination. Only one other study for the combination of A. afra and L. javanica has been researched by van Vuuren (2007). Time-kill assays were utilized to access the ethnobotanical use of A. afra essential oil (0.25%) alone, L. javanica essential oil (0.25%) alone and the 1:1 combination of A. afra (0.125%) with L. javanica (0.125%) essential oils. Time-kill on the two independent plants was carried out on three pathogens i.e. K. pneumoniae, C. neoformans and B. cereus. The time-kill using the combination was carried out against K. pneumoniae. Results on the combination study found that a bactericidal effect was achieved within an hour of exposure and that this cidal effect was maintained for the full 48 hrs of testing. The study concluded that A. afra interacts synergistically with L. javanica against K. pneumoniae thus validating the traditional use of the combination (van Vuuren, 2007) The aims of this study included studying the essential oil composition of A. afra and L. javanica using GC-MS, TLC screening on the plants essential oils, dichloromethane: methanol extracts and the aqueous extracts independently and in combination (1:1) and testing of the antimicrobial activity, of A. afra and L. javanica independently as well as in combination using MIC microplate assays. Combinations were made by mixing different 63 ratios of A. afra with L. javanica. Nine ratios in total were carried out with either A. afra or L. javanica varying in mixed ratios. Additionally, not only the essential oils, but also the dichloromethane: methanol extracts as well as the aqueous extracts were tested. ?FIC determination as well as isobologram studies were then done in order to determine the efficacy of A. afra with L. javanica in combination and identify any interaction between the two. 3.2 Results and discussion 3.2.1 Chromatographic techniques 3.2.1.1 Essential oil composition of A. afra Thirty compounds were identified, making up 92.8% of the total composition (Table 3.1) in the essential oil of A. afra. The major compounds present were camphene (4.0%), 1, 8- cineole (17.0%), artemisia ketone (6.4%), ?-thujone (6.0%), camphor (41.0%) and borneol (8.6%) Table 3.1 GC-MS results of A. afra. RRI* Compounds Area (%) 1016 ?-Pinene 0.5 1057 Camphene 4.0 1104 ?-Pinene 0.4 1193 ?-Terpinene 0.1 1202 1,8-Cineole 17.0 1232 (Z)-?-Ocimene 0.4 1242 ?-Terpinene 0.1 1250 (E)-?-Ocimene 0.1 1281 Terpinolene 0.1 1350 Artemisia ketone 6.4 1441 ?-Thujone 6.0 1488 ?-Copaene 0.1 1501 Artemisia alcohol 0.2 1511 Chrysantenone 0.1 1521 Camphor 41.0 1573 Pinocarvone 0.5 1588 Bornyl acetate 0.5 1602 Terpinen-4-ol 1.7 1628 cis-p-Menth-2-en-1-ol 0.5 1639 Myrtenal 0.5 1653 trans Pinocarveol 1.0 1672 ?-Terpineol 0.2 1679 trans Piperitol 0.3 1701 ?-Terpineol 0.3 64 *RRI: Relative retention indices calculated against n-alkanes. % calculated from TIC data. Numerous studies have reported on the composition of the essential oil of A. afra (Appendix A). The sample of A. afra from this study is rich in camphor at 41.0% and ? ? thujone makes up only 6.0% of the total composition of the oil and ?- thujone was absent. In the study by van Vuuren (2007), the essential oil of A. afra was collected from Klipriviersberg (the same location as this sample) and analysed. The major compounds detected in this study and that of van Vuuren (2007) were mostly the same except ?-thujone (18.8%) was the major compound in van Vuuren (2007) and camphor (41.0%) was the major constituent in this study. As discussed in Section 3.4.2, the differences in the major compound composition of the oils are clearly noticed. This is not surprising, as studies conducted have concluded that A. afra shows immense chemical variation from individual plant-to-plant samples to collective plant samples (Graven et al., 1992; Chagonda et al., 1999; Mangena and Muyima, 1999; Viljoen et al., 2006b). 3.2.1.2 Essential oil composition of L. javanica The GC-MS results for L. javanica showed twenty-one compounds (Table 3.2) making up 96.3%. The major compounds are 1, 8-cineole (2.3%), p-cymene (3.4%), trans-linalool oxide (4.5%), cis-linalool oxide (4.0%), linalool (70.7%) and caryophyllene oxide (6.9%). Viljoen et al. (2005) as well as van Vuuren (2007) studied the chemical composition of L. javanica essential oil from Fairlands in the North of Johannesburg, the same locality as the L. javanica used in this study. Adequate comparison of the differences in the major compounds can thus be made. In Viljoen et al. (2005), linalool (65.19%) was found to be the major compound followed by (Z)-?-ocimene (12.97%) and (E)-?-ocimene (6.21%). van Vuuren (2007) found the major compounds to be (Z)-?-ocimene (13.0%) and linalool (65.2%). Comparing these samples with that of L. javanica essential oil in this study, it is clear that RRI* Compounds Area (%) 1709 Borneol 8.6 1715 Germacrene D 0.9 1739 Piperitone 0.3 1741 Bicyclogermacrene 0.4 1745 Piperitol 0.3 1857 Myrtenol 0.3 Total 92.8 65 linalool is the main or dominant compound, with the composition of linalool alone in all three samples greater than 65.0%. A slight difference that is noted is the absence of (Z)-?-ocimene in the sample tested in this study. In van Vuuren (2007), (Z)-?-ocimene was only found as a major constituent in the Fairland population and was not a main constituent in the other populations tested. Thus, the absence of this constituent, in the sample used in this study, is notable although variations may exist between and within natural populations (Viljoen et al., 2005). Table 3.2 GC-MS results of L. javanica. RRI* Compounds Area (%) 1016 ?-Pinene 0.1 1057 Camphene 0.2 1117 Sabinene 0.1 1193 Limonene 0.5 1202 1,8-Cineole 2.3 1270 p-Cymene 3.4 1441 trans-Linalool oxide 4.5 1464 ?-Longipinene 1.2 1471 cis-Linalool oxide 4.0 1488 ?-Copaene 0.3 1511 Chrysanthenone 0.1 1521 Camphor 0.4 1546 Linalool 70.7 1575 d-Terpineol 0.1 1596 ?-Caryophyllene 0.3 1602 Terpinen-4-ol 0.2 1701 ?-Terpineol 0.1 1709 Borneol 0.8 1728 ?-Amorphene + ?-muurolene 0.1 2010 Caryophyllene oxide 6.9 Total 96.3 *RRI: Relative retention indices calculated against n-alkanes % calculated from TIC data 3.2.1.3 Thin layer chromatography TLC fingerprint studies were carried out as an initial screening process singularly and in combination, in order to determine and visualise the locality of the various compounds by using Rf values. Figure 3.1, 3.2 and 3.3 show respectively the TLC results for essential oils, dichloromethane: methanol extracts and aqueous extracts of A. afra, L. javanica alone and in combination. 66 In Figure 3.1, compounds not visible under UV light of 254 nm or UV of 365 nm or white light are then seen when the TLC plate has been derivatized. Several compounds in A. afra essential oil were detected under 254 nm of UV light. Figure 3.1 TLC of the essential oils of A. afra, L. javanica individually and in combination under UV light of 254 nm, UV light of 365 nm and visualized with vanillin-sulphuric acid reagent. Aa - A. afra; Lj - L. javanica; Aa + Lj -A. afra with L. javanica. The combination of A. afra and L. javanica essential oil showed a mixture of the components of each of the oils, although to a lesser degree. Under UV light of 365 nm, compounds at the starting point for all three lanes were visible. A single compound, red in colour with Rf = 0.35, was visible in L javanica alone, as well as in the combination with A. afra. After derivatization with vanillin-sulphuric acid reagent, many compounds not previously visible were observed. The dichloromethane: methanol extracts (Figure 3.2) showed good separation of the constituents of each plant under UV light of 254 nm. The combination of A. afra with L. javanica also showed very clearly the compounds that are visible singularly. The derivatized plate also demonstrated good separation with a greenish-yellow coloured major compound found in L. javanica (Rf = 0.45). This compound was also clearly visible in the combination wherein A. afra was combined with L. javanica. Aa Lj Aa + Lj Aa Lj Aa + Lj Aa Lj Aa + Lj 254 nm A 365 nm B Derivatized C 67 Figure 3.2 TLC of the dichloromethane: methanol extracts of A. afra, L. javanica individually and in combination under UV light of 254 nm, under UV light of 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa - A. afra; Lj - L. javanica; Aa + Lj -A. afra with L. javanica. The TLC chromatograms of the aqueous extracts (Figure 3.3) of A. afra, L. javanica and their combination showed many compounds at 254 nm and 365 nm. After derivatization, compounds in L. javanica that were present in the dichloromethane: methanol extract were noted in the aqueous extracts of L. javanica and in the combination with A. afra at Rf = 0.45. The HPTLC chromatograms show the complexity of the different plants. This is increased when the plants are combined. Thus, the individual components of the plants singularly and in combination with other plants would be very difficult to identify. The presence of a greater number of bands in the combination TLC shows that combining plants together creates complex phytochemical pools and therefore more possibilities for synergistic interactions between the compounds that are now in contact with each other. There are also increased opportunities for antimicrobial interaction against bacteria as different compounds may inhibit microbes using different mechanisms thus resulting in synergy. Aa Lj Aa + Lj Aa Lj Aa + Lj Aa Lj Aa + Lj Aa Lj Aa +Lj 254 nm A 365 nm B Derivatized C White light 68 Figure 3.3 TLC of the aqueous extracts of A. afra, L. javanica individually and in combination under UV light of 254 nm, under UV light of 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa - A. afra; Lj - L. javanica; Aa + Lj -A. afra with L. javanica. 3.2.2 Antimicrobial analysis 3.2.2.1 MIC assays and FIC determination MIC values obtained for the negative controls, ciprofloxacin and amphotericin B, against the micro-organisms used in the study are shown in Table 3.3. MIC values were interpreted and classified according to Table 2.4, Chapter 2. The negative control, acetone, was noted immediately when checking the results of the experiment and where problems were experienced tests were repeated. Table 3.3 MIC values obtained for controls (?g/ml) Micro-organism Controls Ciprofloxacin/Amphotericin B M. catarrhalis ATCC 23246 6.3 K. pneumoniae NCTC 9633 0.2 E. faecalis ATCC 29212 0.6 C. neoformans ATCC 90112 3.1 S. pneumoniae ATCC 49619 0.3 S. pyogenes ATCC 8668 0.6 S. agalactiae ATCC 55618 1.3 M. smegmatis (clinical) 0.2 Aa Lj Aa + Lj Aa Lj Aa + Lj Aa Lj Aa + Lj Aa Lj Aa +Lj 254 nm A 365 nm B Derivatized C White light 69 Culture purity and signs of contamination were checked by streaking out a loopful of culture onto agar and incubating as necessary and checked immediately after completion of incubation. Where antimicrobial controls did not reflect consistent expected ranges the entire experiment was repeated and the result deleted from the data set. Generally, the MIC values for A. afra and L. javanica were in the moderate range and showed good consistency from sample to sample. 3.2.2.1.1 Essential oil A. afra has a broad spectrum of activity (Mangena and Muyima 1999) The MIC results of the essential oils of A. afra (Table 3.4) showed poor antimicrobial activity against K. pneumoniae, M. catarrhalis and E. faecalis with mean MIC values at 8.0, 8.7 and 8.7 mg/ml respectively. Some antimicrobial activity was seen against C. neoformans (3.8 mg/ml), S. pneumoniae (3.3 mg/ml) and S. agalactiae (4.8 mg/ml). Table 3.4 MIC and ?FIC values of A. afra, L. javanica essential oils alone and in combination against different pathogens (mg/ml). * Values in bold indicate noteworthy activity. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Noteworthy antimicrobial activity was seen against S. pyogenes and M. smegmatis whereby mean MIC values of 1.7 and 1.5 mg/ml were obtained. Thus A. afra essential oil was the Micro-organism MIC (mg/ml) ?FIC A. afra L. javanica A. afra with L. javanica A. afra with L. javanica (1:1) Interpretation K. pneumoniae NCTC 9633 8.0 8.0 8.0 1.0 Additive M. catarrhalis ATCC 23246 8.7 8.0 8.0 1.0 Additive E. faecalis ATCC 29212 8.7 8.0 8.0 1.0 Additive C. neoformans ATCC 90112 3.8 1.5 2.0 0.9 Additive S. pneumoniae ATCC 49619 3.3 2.0 2.0 0.8 Additive S. pyogenes ATCC 8668 1.7 1.4 1.0 0.7 Additive S. agalactiae ATCC 55618 4.8 4.0 4.0 0.9 Additive M. smegmatis (clinical) 1.5 2.0 2.0 1.2 Indifferent 70 most active in inhibiting the growth of M. smegmatis and can be considered a moderate antimicrobial inhibitor. The antimicrobial activity of A. afra essential oil has been widely studied using the disc diffusion method or the MIC micro-titre plate method. In order to further substantiate the results obtained by disk diffusion and the MIC assays, time-kill studies were employed. In a disc diffusion study by Graven et al. (1992), A. afra essential oil was found to be active against Acinetobacter calcoaceticus, Beneckea natriegens, Brevibacterium linens, Citrobacter freundii, Klebsiella pneumoniae, E. faecalis, Moraxella spp. and Serratia marcescens. A. afra also exhibits known fungicidal activity as described in the study by Gundidza (1993). It was shown that the essential oil has a strong inhibitory effect on Candida albicans, Aspergillus niger, Alternia alternate, Aspergillus ochraceus, Geotrichum candidum, Penicillium citrium and Aspergillus parasiticus. A later study by Mangena and Muyima (1999) using the disc diffusion assay on the essential oil of A. afra indicated that it had a broad spectrum antimicrobial activity. Among the most sensitive pathogens to the oil were S. pyogenes, Listeria monocytogenes and Acinetobacter johnsonii. S. pyogenes was used in this study as well and noteworthy antimicrobial activity was also seen with A. afra essential oil with a mean MIC value of 1.7 mg/ml. Due to the different methods used to assess the antimicrobial activity, direct comparisons cannot be made. However, the antimicrobial activity noted in this study and the one by Mangena and Muyima (1999) serves to confirm the inhibitory effect of A. afra on S. pyogenes which is a common cause of respiratory related conditions (Khan et al., 2009b). In 2002, Muyima et al. conducted a study on the antimicrobial activity of A. afra essential oil. Results found using the disc diffusion assay showed a high level of sensitivity of A. afra towards E. coli, S. aureus, Candida albicans and Ralstonia pickettii. Huffman et al. (2002) examined the antimicrobial activity of the essential oil of A. afra using the microplate method and minimum inhibitory percentages were determined. Candida albicans and C. neoformans gave minimum inhibitory percentages of 0.25%. In 2006, van Vuuren and Viljoen conducted a study on the antimicrobial activity of the essential oil of A. afra using the MIC micro-titre plate and the time-kill method. The essential oil was tested against ten pathogens including S. aureus, S. epidermidis, B. cereus, B. subtilis, E. coli, P. 71 aeruginosa, E. faecalis, K. pneumoniae, C. albicans and C. neoformans. A. afra exhibited MIC values of 5.7, 10.1 and 8.0 mg/ml against E. faecalis, K. pneumoniae and C. neoformans, respectively. These results are in congruence with those attained in this current study. Time-kill studies were performed against S. aureus, K. pneumoniae and C. albicans. The time-kill study performed against S. aureus and C. albicans revealed bactericidal efficacy after four hours of exposure to the essential oil of A. afra (0.5%). However, against K. pneumoniae, regrowth was noted after one hour of exposure. In another time-kill study by Viljoen et al. (2006b) carried out on A. afra essential oil, A. afra was tested against K. pneumoniae and C. neoformans over time to determine if a bactericidal effect was apparent in order to validate the traditional use for respiratory conditions. Against K. pneumoniae a bactericidal effect was noted at 0.75% within 10 min of the test. A bactericidal effect was noted for 1.0% of A. afra against C. neoformans within 60 min. Thus, the traditional use of A. afra essential oil was validated for the treatment of respiratory conditions. It is important to note that A. afra essential oil, to my knowledge, has never been tested before using the MIC micro-titre plate method against S. pyogenes, S. agalactiae and S. pneumoniae. This is somewhat surprising as these are key pathogens in the causes of respiratory conditions in South Africa and in the world. Furthermore, activities found for S. pyogenes were one of the best of all pathogens tested. L. javanica essential oil showed poor antimicrobial activity against K. pneumoniae, M. catarrhalis and E. faecalis with mean MIC values of 8.0 mg/ml. Against S. agalactiae, some antimicrobial activity was noted with a mean MIC value of 4.0 mg/ml. Against S. pneumoniae and M. smegmatis, noteworthy activity was seen, both giving a mean MIC value of 2.0 mg/ml. The best antimicrobial activity of L. javanica essential oil was seen against C. neoformans and S. pyogenes that gave mean MIC values of 1.5 and 1.4 mg/ml respectively. L. javanica essential oil can be further classified as a moderate inhibitor of C. neoformans and S. pyogenes. The noteworthy antimicrobial activity seen against C. neoformans, S. pneumoniae, S. pyogenes and M. smegmatis therefore supports the traditional use of L. javanica. 72 Antimicrobial efficacies for L. javanica essential oil has been demonstrated against E. coli, S. aureus, Salmonella gallnarum, Klebsiella pneumoniae, Candida albicans P. aeruginosa, S. epidermidis, E. cloacae (Mwangi et al., 1992; Chagonda et al., 1993; Ngassapa et al., 2003; Subramoney, 2003; Manenzhe et al., 2004). Samie et al. (2005) conducted a study testing the essential oil of L. javanica using the disc diffusions assay as well as the micro-dilution method. Disk diffusion showed activity against B. pumilus, B. subtilis, E. faecalis, E. cloacae, E. coli, P. mirabilis and K. pneumoniae. The micro-dilution method revealed activity against B. cereus, B. pumilus, B. subtilis, S. aureus, E. faecalis, E. cloacae, E. coli, P. agglomerans, P. aeruginosa, S. flexneri, A. hydrophila P. mirabilis, K. pneumoniae and S. cholera-suis. The MIC values obtained by Samie et al. (2005) against E. faecalis and K. pneumoniae were 3.0 mg/ml. These were lower than that noted in this current study where MIC values of 8.0 mg/ml were noted. Different geographical locations for collection of L. javanica could be the reason behind the differences in MIC values. It is known that L. javanica essential oil exhibits chemical variation with respect to geographical distribution (Viljoen et al., 2005) and this may contribute to the varied antimicrobial effects noted. Time-kill studies by Viljoen et al. (2005) against K. pneumoniae showed bactericidal effect within 30 min for all the concentrations tested. Against C. neoformans, the killing rate at 1% concentration was within one hour for L. javanica. No bactericidal effect was noted against B. cereus over a 24 hr period. These results validated the use of L. javanica in African traditional healing to treat the symptoms of colds and flu (Viljoen et al., 2005). van Vuuren and Viljoen (2006) tested the antimicrobial activity of L. javanica essential oil from the same locality against S. aureus, S. epidermidis, B. cereus, B. subtilis, E. coli, P. aeruginosa, E. faecalis, K. pneumoniae, C. albicans and C. neoformans. MIC values obtained against E. faecalis were 13.5 mg/ml, K. pneumoniae, 7.6 mg/ml and C. neoformans, 2.4 mg/ml. Congruency between results of the present study was noted. A time-kill study was also performed by van Vuuren and Viljoen (2006) using 0.5% essential oil of L. javanica against S. aureus, K. pneumoniae and C. albicans. Against S. aureus a 50% reduction in CFU/ml was noted over 24 hrs however, no bactericidal activity was noted against K. pneumoniae. Against C. albicans, bactericidal activity was only noted after 24 hrs of exposure. These differences in activities seen between Viljoen et al. (2005), and van Vuuren 73 and Viljoen (2006), wherein the samples of L. javanica was from the same locality, are possibly due to the qualitative and quantitative variation seen between and within the populations of L. javanica. These variations occur where even a minor compound could affect the antimicrobial activity of the plant essential oil. This demonstrates that the possibilities of synergistic, antagonistic or even additive interactions are not to be ignored (Nakatsu et al., 2000; van Vuuren and Viljoen, 2006). The combination of A. afra with L. javanica essential oils showed poor antimicrobial activity against K. pneumoniae, M. catarrhalis and E. faecalis giving mean MIC values of 8.0 mg/ml. No increase in the MIC value, as compared to the individual oils, was noted. Some antimicrobial activity was seen against S. agalactiae with a mean MIC value of 4.0 mg/ml. Noteworthy antimicrobial activity was noted against C. neoformans, S. pneumoniae and M. smegmatis with mean MIC values of 2.0 mg/ml. However, against S. pyogenes a mean MIC value of 1.0 mg/ml was noted. The combination is thus classified as a moderate inhibitor of S. pyogenes. This was the most sensitive pathogen to the combination of the essential oils of A. afra with L. javanica. ?FIC values calculated from the combination of A. afra with L. javanica essential oil against K. pneumoniae, M. catarrhalis, E. faecalis, C. neoformans, S. pneumoniae, S. pyogenes and S. agalactiae ranged from 0.7-1.0. This indicates that the combination showed additive interactions against these pathogens. Only one pathogen i.e. M. smegmatis showed indifferent interactions for the combination with a ?FIC value of 1.2. It is important to note that no antagonism against the pathogens tested was noted for the combination. The combination of A. afra with L. javanica essential oils has been tested against one pathogen using a time kill assay (van Vuuren, 2007), as mentioned in Section 3.1. There have been numerous citations on the use of the combination in traditional African medicine (Smith, 1895; Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; Neuwinger, 2000; Huffman et al., 2002). L. javanica is cited to be used in combination with Aloysia triphylla as an herbal tea for the treatment of colds, fever and asthma (van Wyk and Wink. 2004). However, scientific validation of the efficacy of the combination is nowhere to be found. Even though van Vuuren (2007) has studied the combination, different methodologies were not used in order to assess the presence of various interactions and against different micro- 74 organisms. Since no previous studies on the combination have been done, comparisons cannot be made. However, it should be noted that no antagonism was seen in the combination against any of the pathogens tested. Mostly additive interactions were prominent and where additivity was absent, indifference was evident. Further interactive efficacies are elaborated on in Section 3.2.2.2. 3.2.2.1.2 Dichloromethane: methanol extracts The MIC values observed for the dichloromethane: methanol extracts, as shown in Table 3.5, of A. afra showed some antimicrobial activity against K. pneumoniae (6.3 mg/ml), M. catarrhalis (4.7 mg/ml), E. faecalis (6.3 mg/ml), C. neoformans (1.2 mg/ml), S. pyogenes (1.1 mg/ml), S. agalactiae (3.0 mg/ml) and M. smegmatis (1.7 mg/ml). The antimicrobial activity of A. afra against C. neoformans and S. pyogenes (MIC values of 1.2 and 1.1 mg/ml) can further classify A. afra dichloromethane: methanol extract as a moderate inhibitor of these two pathogens. Noteworthy and probably the best antimicrobial activity were seen for A. afra against S. pneumoniae where a mean MIC value of 0.5 mg/ml was obtained. A. afra dichloromethane: methanol extract can thus be further classified as being a strong inhibitor of S. pneumoniae. A. afra methanol extracts were tested for antimicrobial activity using the disc diffusion assay by Rabe and van Staden (1997) and activity was noted against S. aureus and B. subtilis. McGaw et al. (2000) later performed disc diffusion assays as well as MIC dilution assays to determine the antimicrobial activity of A. afra. The hexane extract and the ethanol extracts were tested. No activity against the pathogens at the concentration tested (12.5 mg/ml) was seen with the hexane extracts. The ethanol extracts however, showed activity against B. subtilis and S. aureus when using both antimicrobial assays. Braithwaite et al. (2008) studied the methanol and the acetone extracts of A. afra to validate smoke inhalation therapy to treat some microbial infections. The MIC micro-titre plate assay was used and noteworthy activity (<1.0 mg/ml) was seen only for the acetone extracts against S. aureus (MIC value = 0.25 mg/ml), B. cereus (MIC value = 0.25 mg/ml) and C. neoformans (MIC value = 0.75 mg/ml). Some antimicrobial activity was noted with the acetone extract of A. afra against K. pneumoniae with an MIC value of 2.0 mg/ml. Results obtained in this study vary slightly compared to previous studies mentioned. However, direct comparisons are difficult as the type of extracts and the pathogens tested are different. 75 Table 3.5 MIC and ?FIC values of A. afra and L. javanica dichloromethane: methanol extracts alone and in combination against different pathogens (mg/ml). Micro-organism MIC (mg/ml) ?FIC A. afra L. javanica A. afra with L. javanica A. afra with L. javanica (1:1) Interpretation K. pneumoniae NCTC 9633 6.3 16.0 6.0 0.7 Additive M. catarrhalis ATCC 23246 4.7 6.0 6.0 1.1 Indifferent E. faecalis ATCC 29212 6.3 2.5 4.7 1.3 Indifferent C. neoformans ATCC 90112 1.2 3.5 2.0 1.1 Indifferent S. pneumoniae ATCC 49619 0.5 0.4 0.6 1.4 Indifferent S. pyogenes ATCC 8668 1.1 0.4 1.5 2.6 Indifferent S. agalactiae ATCC 55618 3.0 2.2 1.1 0.4 Synergy M. smegmatis (clinical) 1.7 1.5 2.5 1.6 Indifferent * Values in bold indicate noteworthy activity. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. When Mativandlela et al. (2008) tested the ethanol extracts of A. afra against M. smegmatis; an MIC value of 1.56 mg/ml was obtained. This result is in congruence with the findings in this study where the mean MIC value obtained against M. smegmatis was 1.7 mg/ml. This report by Mativandlela et al. (2008) together with the results from this study provides evidence to support the use of A. afra against certain respiratory pathogens. L. javanica dichloromethane: methanol extract displayed very poor antimicrobial activity against K. pneumoniae with a mean MIC value of 16.0 mg/ml. MIC values ranging from 2.2 - 6.0 mg/ml, showing some antimicrobial activity, were obtained against S. agalactiae, E. faecalis, C. neoformans and M. catarrhalis. Moderate inhibition was obtained against M. smegmatis with a MIC value of 1.5 mg/ml. Noteworthy activity with strong inhibition was noted for L. javanica against S. pneumoniae and S. pyogenes with mean MIC values of 0.4 mg/ml. 76 L. javanica methanol, acetone and hexane extracts have been tested for their antimicrobial activity (Samie et al., 2005) using the disc diffusion and the MIC micro-titre plate method. The methanol extract of the leaves were active against B. cereus, B. subtilis, E. faecalis, E. cloacae, E. coli, S. flexneri, A. hydrophila and S. cholera-suis with MIC values ranging between 1.5-6.0 mg/ml. Against K. pneumoniae poor activity was noted with an MIC value of 12.0 mg/ml. Against E. faecalis, some antimicrobial activity was evident where a MIC value of 3.0 mg/ml was noted. This correlates with the results found in this study wherein MIC values against K. pneumoniae and E. faecalis were 16.0 and 2.5 mg/ml respectively. The acetone extracts exhibited similar activities to the methanol extracts with MIC values of 6.0 mg/ml against E. faecalis and 12.0 mg/ml against K. pneumoniae. The hexane extracts exhibited less activity against the tested pathogens with poor antimicrobial activity against E. faecalis and K. pneumoniae (12.0 mg/ml). The methanol extracts of L. javanica were similarly tested against four pathogens i.e. S. aureus, E. faecalis, E. coli and P. aeruginosa (Shikanga et al., 2010). Noteworthy activities were noted with all the MIC values below 1.0 mg/ml. Relevant to the current study, was the MIC value obtained against E. faecalis of 0.14 mg/ml. This denoted very strong inhibition and very interesting activity. However, this was not a result that was demonstrated in the current study where the MIC value obtained was 2.5 mg/ml. This difference could be due to the differing collection localities and/or the chemical variations between and in natural populations (Viljoen et al., 2005). Samie at el. (2010) tested the acetone and the hexane extracts of the leaves of L. javanica against three fungal organisms. Antifungal activity was noted for the acetone extracts against Candida krusei (MIC value of 1.88 mg/ml). The hexane extracts showed activity against C. albicans and C. krusei, both with MIC values of 3.75 mg/ml. No activity against C. neoformans (MIC value of >7.5 mg/ml) at the highest concentration tested was noted for either the acetone or the hexane extracts. However, in the current study some activity (MIC value = 3.5 mg/ml) was noted against C. neoformans. Again, geographical and chemical variations may be a contributing factor. L. javanica dichloromethane: methanol extracts have not previously been tested against S. pneumoniae and S. pyogenes (where best activities were noted in this study). This is 77 surprising as Streptococcus spp are well known respiratory organisms and the traditional use of L. javanica is for the treatment of just such infections. The results obtained in this study, however, serve to validate the use of L. javanica in the treatment of S. pneumoniae (bacterial pneumoniae) and S. pyogenes (?strep? throat) related infections. The combination of A. afra with L. javanica dichloromethane: methanol extracts showed some antimicrobial activity against K. pneumoniae, M. catarrhalis, E. faecalis, C. neoformans and M. smegmatis with MIC values ranging from 2.0-6.0 mg/ml. Moderate inhibition was noted against S. pyogenes and S. agalactiae. The best antimicrobial activity with noteworthy inhibition was found against S. pneumoniae with an MIC value of 0.6 mg/ml. ?FIC values calculated showed indifferent interactions for all the pathogens tested with the exception of K. pneumoniae and S. agalactiae. An additive interaction was noted against K. pneumoniae with a ?FIC value of 0.7. A synergistic interaction of the combination of A. afra with L. javanica was seen with ?FIC = 0.4 against S. agalactiae. The particular combination of A. afra with L. javanica dichloromethane: methanol extracts have also not been studied previously. From this study, the ?FIC results did not show any antagonistic interactions. In fact, additivity was noted against K. pneumoniae (?FIC value of 0.7). Synergy was observed for S. agalactiae (?FIC value of 0.4). As K. pneumoniae is a leading cause of nosocomial pneumoniae (Ko et al., 2002; Yu et al., 2007; Brisse et al., 2009) in South Africa; this may prove to be a useful discovery. 3.2.2.1.3 Aqueous extracts The aqueous extracts of A. afra (Table 3.6) showed generally poor antimicrobial activity against the pathogens tested with some antimicrobial activity against C. neoformans (4.0 mg/ml) and S. pyogenes (5.3 mg/ml). The best activity of A. afra aqueous extract was seen against S. agalactiae giving weak antimicrobial activity with a mean MIC value of 1.7 mg/ml. poor antimicrobial activity was noted against M. catarrhalis, S. pneumoniae and M. smegmatis, all yielding mean MIC values of 8.0 mg/ml while against E. faecalis a MIC value of 7.0 mg/ml was obtained. Very poor activity was seen against K. pneumoniae where a mean MIC value of 12.0 mg/ml was noted. 78 Table 3.6 MIC and ?FIC values of A. afra, L. javanica aqueous extracts alone and in combination against different pathogens (mg/ml). * Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for L. javanica was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. The aqueous extracts of A. afra were tested by Rabe and van Staden (1997) and no activity at the concentration tested (100.0 mg/ml) was noted when using the disc diffusion method for antimicrobial assessment. McGaw et al. (2000) also tested A. afra aqueous extracts and found no antimicrobial activity using the disc diffusion method as well as the MIC micro-titre plate method. Aqueous extracts have been known to exhibit poor to no antimicrobial activity (J?ger, 2003; Abu-Shanab et al., 2004; van Vuuren, 2008). This is in contrast to the traditional method of preparation of plants where most often the infusion or the decoctions are used. L. javanica aqueous extract showed some antimicrobial activity against C. neoformans, S. pneumoniae, S. pyogenes and M. smegmatis with mean MIC values ranging between 3.0-6.0 mg/ml. Against K. pneumoniae and M. catarrhalis, L. javanica was not active at the highest Micro-organism MIC (mg/ml) ?FIC A. afra L. javanica A. afra with L. javanica A. afra with L. javanica (1:1) Interpretation K. pneumoniae NCTC 9633 12.0 ?16.0 12.0 ND1 Additive2 M. catarrhalis ATCC 23246 8.0 ?16.0 8.0 ND1 Additive2 E. faecalis ATCC 29212 7.0 12.0 8.0 0.9 Additive C. neoformans ATCC 90112 4.0 3.5 3.0 0.8 Additive S. pneumoniae ATCC 49619 8.0 4.0 8.0 1.5 Indifferent S. pyogenes ATCC 8668 5.3 3.0 4.0 1.0 Additive S. agalactiae ATCC 55618 1.7 0.5 0.8 1.0 Additive M. smegmatis (clinical) 8.0 6.0 8.0 1.2 Indifferent 79 concentration tested while very poor activity was seen against E. faecalis with a mean MIC value of 12.0 mg/ml. Interestingly, noteworthy strong antimicrobial inhibition was noted against S. agalactiae where a mean MIC value of 0.5 mg/ml was obtained. Thembo et al. (2010) tested the aqueous extracts of L. javanica and found no activity against some mycotoxigenic fungi. However, results from the current study provided an exception against S. agalactiae wherein strong inhibitory activity (MIC value of 0.5 mg/ml) was noted. S. agalactiae is a respiratory tract pathogen and causes pneumonia in healthy humans. The use of L. javanica infusions and decoctions in traditional medicine may have solid scientific support due to the inhibition observed in this study. Shikanga et al. (2010) tested the methanol extracts of L. javanica as a substitute of the aqueous extracts as the extract compositions were similar and the methanol extracts are easier to dry. The study was performed to determine the antioxidant and antibacterial activity of L. javanica infusions. Results were promising with noteworthy activities recorded. When A. afra was combined with L. javanica aqueous extract very poor antimicrobial activity was noted against the pathogen K. pneumoniae (MIC value of 12.0 mg/ml) and low activity was noted against M. catarrhalis, E. faecalis, S. pneumoniae and M. smegmatis (mean MIC values of 8.0 mg/ml). Some antimicrobial activity was seen against C. neoformans and S. pyogenes with mean MIC values of 3.0 and 4.0 mg/ml. Noteworthy activity was again noted against S. agalactiae, with a mean MIC value of 0.8 mg/ml. ?FIC values for the combination of A. afra with L. javanica against K. pneumoniae and M. catarrhalis could not be calculated, as the end point MIC value of L. javanica was not determined. However, based on the MIC values a tentative interpretation is made. Thus against K. pneumoniae and M. catarrhalis the combination of A. afra with L. javanica aqueous extracts additive interactions are apparent. Additivity is also evident against E. faecalis, C. neoformans, S. pyogenes and S. agalactiae from the calculated ?FIC values. Indifferent interactions were noted against S. pneumoniae and M. smegmatis. The combination of A. afra with L. javanica aqueous extract has not been studied previously. In this study, low to poor activity was noted when testing this combination. ?FIC values showed the combination was additive for most of the pathogens while some were indifferent. Interestingly S. agalactiae was the most sensitive pathogen to the combination. Thus, this 80 combination would be ideal in the treatment of S. agalactiae and S. pyogenes related respiratory infections. 3.2.2.2 Isobologram interpretation A. afra, tested with L. javanica essential oils in combination against M. catarrhalis at different ratios were plotted on isobolograms. Results display additive interactions with all points on the line (Figure 3.4). The dichloromethane: methanol extracts of the same combination also yield additive interactions with two exceptions. The exceptions, are two points (ratios 2:8 (Figure 3.4, point a) and 1:9 (Figure 3.4, point b), below ?FIC = 0.5, wherein L. javanica is in the majority. These two combinations display synergistic interactions. The 1:1 ratio of the combination is represented by a square ( ) in the isobologram. Figure 3.4 Isobologram of the combination of A. afra with L. javanica essential oils ( ) and the dichloromethane: methanol extracts ( ) against M. catarrhalis (a ? ratio 2:8; b ? ratio 1:9); ? 1:1 combination. The aqueous extracts have not been included in the isobologram as the end point for L. javanica was not determined at the highest concentration tested, thus an isobologram could not be constructed. However, a bar graph as depicted in Figure 3.6 shows the MIC values of the different ratios compared to the MIC values of A. afra independently (8.0 mg/ml) and L. javanica (?16.0 mg/ml) alone against M. catarrhalis. The MIC values of the combination of 0.00 0.25 0.50 0.75 1.00 1.25 1.50 0.00 0.25 0.50 0.75 1.00 1.25 1.50 M. catarrhalis ATCC 23246 MIC Aa in combination/MIC Aa independently M IC Lj in co m bi na ti on /M IC Lj in de pe nd en tl y a b 81 A. afra with L. javanica at ratios 9:1 and 8:2 were consistent at 8.0 mg/ml. At the ratio of 7:3, the MIC value went up to 16.0 mg/ml and at 6:4, the MIC value noted was 12.0 mg/ml. Against K. pneumoniae (Figure 3.5) the combination of A. afra with L. javanica essential oils yielded additive interactions for all the ratios tested. The dichloromethane: methanol extracts of the combination showed additive interactions for all ratios. The aqueous extracts could not be included in the isobologram as the end point of L. javanica could not be determined. Figure 3.5 Isobologram of the combination of A. afra with L. javanica essential oils ( ) and the dichloromethane: methanol extracts ( ) against K. pneumoniae; ? 1:1 combination. The bar graph of the MIC values obtained for the different ratios tested are shown in Figure 3.6. This shows that at higher concentrations of A. afra, between ratios 9:1, 8:2 and 6:4, the MIC values (6.0 mg/ml) of the combination is lower than that of A. afra alone or L. javanica alone. The MIC value of the ratio 7:3 was the same as A. afra, 8.0 mg/ml. When combined in equal ratios, the activity of the combination is the same as that of A. afra alone. However, when L. javanica is in the majority, the MIC of the combination was higher than the concentration of the combination that was tested i.e. ?16.0 mg/ml. Where A. afra (aqueous extract) is present in higher concentrations, better antimicrobial activity is noted against K. pneumoniae (Figure 3.6). 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 K. pneumoniae NCTC 9633 MIC Aa in combination/MIC Aa independently M IC Lj in co m bi na ti on /M IC Lj in de pe nd en tl y 82 Figure 3.6 The MIC values of the different ratios of the combination of A. afra with L. javanica aqueous extracts against M. catarrhalis and K. pneumoniae. In the combination of A. afra with L. javanica against E. faecalis (Figure 3.7), the essential oils showed additive pharmacological interaction for all the ratios tested. The dichloromethane: methanol extracts showed additive interactions as well for most of the ratios with the exception of two ratios. The ratio 2:8 (Figure 3.7, point c) showed additivity and the ratio 1:9 (Figure 3.7, point d) was synergistic. This suggests that an ideal combination of A. afra with L. javanica dichloromethane: methanol extracts would be with one part of A. afra and nine parts of L. javanica. The aqueous extracts showed additive interactions for all the ratios tested with one ratio i.e. 9:1, was found on the 1:0 and 0:1 line of the isobologram. A. afra was the majority plant in this combination. Isobologram interactions of the combination of A. afra with L. javanica essential oils against C. neoformans (Figure 3.8) resulted in additive interactions. The dichloromethane: methanol extract also gave additivity at the ratios tested. Two ratios however, showed indifference when combined. These were the ratios 7:3 (Figure 3.8, point e), where A. afra was in the majority and 1:9 (Figure 3.8, point f), where L. javanica was dominant. When the aqueous 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10 M IC val u e (m g/ m l) Ratio (A. afra: L. javanica) M. catarrhalis K. pneumoniae 83 extracts of A. afra and L. javanica were combined in nine ratios, additive interactions were also noted Figure 3.7 Isobologram of the combination of A. afra with L. javanica essential oils ( ), dichloromethane: methanol ( ) and aqueous extracts ( ) against E. faecalis (c ? ratio 2:8; d ? ratio 1:9); ? 1:1 combination. Figure 3.8 Isobologram of the combination of A. afra with L. javanica essential oils ( ) dichloromethane: methanol ( ) and aqueous extracts ( ) against C. neoformans (e ? ratio 7:3; f ? ratio 1:9); ? 1:1 combination. The 1:1 combination of A. afra with L. javanica essential oil and dichloromethane: methanol extracts of the isobolograms (Figure 3.4 and 3.5) and the ?FIC values showed good 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 E. faecalis ATCC 29212 MIC Aa in combination/MIC Aa independently M IC Lj in co m bi na ti on /M IC Lj in de pe nd en tl y c d 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 .75 1.00 1.25 C. neoformans ATCC 90112 MIC Aa in combination/MIC Aa independently M IC Lj in co m bi na ti on /M IC Lj in de pe nd en tl y e f 84 correlation and congruency against M. catarrhalis and K. pneumoniae (Table 3.4 and 3.5). Against E. faecalis and C. neoformans, similarities were apparent for the combination of the essential oils and the aqueous extracts. Partial congruency was noted with the differences in the dichloromethane: methanol extracts where additivity was seen in the isobolograms and indifference was seen in the ?FIC values. One possible explanation for this variability is the possibility of disparate mechanisms of interaction due to the differing methods of testing (Beale and Sutherland, 1983; van Vuuren, 2007). 3.3 Conclusions ? The major constituents of the essential oil of A. afra were camphene (4.0%), 1,8- cineole (17.0%), artemisia ketone (6.4%), ?-thujone (6.0%), camphor (41.0%) and borneol (8.6%), representing 83.0% accumulatively. ? The major compounds of the essential oil of L. javanica are 1,8-cineole (2.3%), p- cymene (3.4%), trans-linalool oxide (4.5%), cis-linalool oxide (4.0%), linalool (70.7%) and caryophyllene oxide (6.9%), representing accumulatively 91.8%. ? The TLC studies for A. afra and L. javanica essential oils, dichloromethane: methanol extracts and the aqueous extracts showed visible bands under different wavelengths and gave a unique fingerprint of the plants independently and in combination used in this assay. ? The dichloromethane: methanol extracts of A. afra indicated better antimicrobial activity (MIC values between 0.5-6.3 mg/ml) than that of A. afra essential oil (MIC values between 1.5-8.7 mg/ml) followed by the aqueous extracts with MIC values ranged from 1.7-12.0 mg/ml. The best activity for the dichloromethane: methanol extracts of A. afra was against S. pneumoniae with a MIC value of 0.5 mg/ml. ? The highest efficacy, using the ?FIC determination method, in the combination study of A. afra with L. javanica was noted with the dichloromethane: methanol extracts against S. agalactiae (?FIC value of 0.4) which was synergistic. ? Synergistic interactions in the isobologram study of the combination of A. afra with L. javanica were noted with the best activity noted in two ratios for the dichloromethane: methanol extracts against M. catarrhalis. One ratio against E. faecalis was also found to be synergistic ? No antagonism for the combination was noted against any of the pathogens tested. 85 ? The efficacies shown against the pathogens tested, using the MIC micro-titre plate, ?FIC determination and the isobologram methods give credence and provide evidence to support the traditional use of A. afra with L. javanica in combination for the treatment of M. catarrhalis, E. faecalis, and S. agalactiae related respiratory tract infections. It should, however, be noted that further toxicity and in vivo studies are needed to confirm safety and efficacy. 86 Chapter 4 The antimicrobial efficacy of Artemisia afra Jacq. Ex Willd and Osmitopsis asteriscoides (L.) Cass. In combination. 4.1 Introduction The plants A. afra and O. asteriscoides have been mentioned in the ethnobotanical literature for their use in combination. Traditionally, a tincture of this combination has been used for the treatment of respiratory complaints (Watt and Breyer-Brandewijk, 1962). The aim of the study of this combination was to determine if any increase, decrease or non-inhibitory effect was noted when the two plants were combined. A monograph (medicinal uses, botanical description, geographical distribution and locality) for O. asteriscoides can be found in Appendix A7. 4.2 Results and discussion 4.2.1 Chromatographic techniques 4.2.1.1 Essential oil composition of O. asteriscoides GC-MS results revealed a total of 32 compounds with the major compound as 1,8-cineole (59.0%). As shown in Table 4.1, camphor and ?-terpineol were also present at 8.2 and 7.2% respectively. The major compounds accumulatively make up 74.4% of the total composition of the oil (91.6%). In the study by Viljoen et al. (2003), where O. asteriscoides was collected from a population near Betty?s Bay in the South Western Cape region of South Africa, the two major compounds found in the pooled sample of plants from the population were 1,8-cineole and camphor, representing 59.9% and 12.4% respectively of the total oil. Other minor compounds were also found. These included ?-pinene, camphene, ?-cymene, longipinene, and ?- terpineol. In the current study the percentage of 1,8-cineole is congruent (59.0%) with that of Viljoen et al. (2003) and cited in van Vuuren and Viljoen (2006). The percentage of ?- terpineol in this study (7.2%) is congruent with that of Viljoen et al, (2003) which contained 7.8% of the total essential oil. The percentage of camphor (8.2%) is slightly different to 87 Viljoen et al. (2003) which made up 12.4% of the total composition of oil. These differences in chemical composition are most likely due to the different collection sites. Table 4.1 GC-MS profile of O. asteriscoides. RRI* Compounds Area (%) 1016 ?-Pinene 2.0 1019 ?-Thujene 0.1 1057 Camphene 1.1 1104 ?-Pinene 0.5 1117 Sabinene 1.2 1159 Myrcene 0.1 1193 ?-Terpinene 0.3 1184 2,3-Dehydro-1,8-cineole 0.2 1193 Limonene 0.1 1202 1,8-Cineole 59.0 1242 ?-Terpinene 0.5 1270 p-Cymene 0.7 1281 Terpinolene 0.1 1382 (z)-Hex-3-en-1-ol 0.1 1441 trans-Linalool oxide 0.1 1456 trans Sabinene hydrate 0.2 1464 ?-Longipinene 2.7 1479 ?-Ylangene 0.1 1511 Chrysanthenone 0.4 1521 Camphor 8.2 1546 Linalool 0.4 1562 Terpineol-4-ol-acetate 0.2 1573 Pinocarvone 0.2 1586 ?-Elemene 0.1 1602 Terpinen-4-ol 2.3 1675 d-Terpineol 0.4 1701 ?-Terpineol 7.2 1709 Borneol 1.1 1745 Piperitol 0.1 1876 cis Carveol 0.1 2010 Caryophyllene oxide 0.4 2122 ?-Bicyclofarnesol 1.4 Total 91.6 *RRI: Relative retention indices calculated against n-alkanes. % calculated from TIC data. 4.2.1.2 Thin layer chromatography The thin layer chromatography of the essential oils (Figure 4.1) of O. asteriscoides individually (Figure 4.1, lane 2) and the combination with A. afra (Figure 4.1, Lane 3) at 254 nm showed some polar compounds. Compounds in A. afra alone (Figure 4.1, lane 1) are 88 visible in the combination as well. After derivatization, many more compounds were visible. Major bands at Rf = 0.2, 0.25, 0.4 and 0.45 were evident. Figure 4.1 TLC of the essential oils of A. afra, O. asteriscoides individually and in combination at 254 nm, 365 nm and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Oa ? O. asteriscoides; Aa + Oa ? A. afra with O. asteriscoides. The dichloromethane: methanol extracts showed that good separation was achieved as shown in Figure 4.2. Many compounds were visible under 254 nm. Under 365 nm, the highly fluorescent compounds at Rf = 0.3 and 0.35 (blue in colour) found in A. afra were also visible. Figure 4.2 TLC of the dichloromethane: methanol extracts of A. afra, O. asteriscoides individually and in combination 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Oa ? O. asteriscoides; Aa + Oa ? A. afra with O. asteriscoides. 254 nm A 365 nm B White light C Derivatized D Aa Oa Aa + Oa Aa Oa Aa + Oa Aa Oa Aa + Oa Aa Oa Aa + Oa 254 nm A 365 nm B Derivatized C Aa Oa Aa + Oa Aa Oa Aa + Oa Aa Oa Aa + Oa 89 Scott et al. (2004), studied the dichloromethane extracts of O. asteriscoides and found four bands when derivatized at Rf = 0.25 (yellow), 0.39 (yellow), 0.58 (purple) and 0.73 (light purple. Similar bands were detected in this study with an extra major band at Rf = 0.8. The aqueous extracts showed poor separation for O. asteriscoides and the combination with A. afra. A major band was visualized after derivatization at Rf = 0.9 for O. asteriscoides and the combination. Figure 4.3 TLC of the aqueous extracts of A. afra, O. asteriscoides individually and in combination at 254 nm; 365 nm; visualized under white light; visualized with vanillin- sulphuric acid reagent. Aa ? A. afra; Oa ? O. asteriscoides; Aa + Oa ? A. afra with O. asteriscoides. 4.2.2 Antimicrobial analysis 4.2.2.1 MIC assays and FIC determination 4.2.2.1.1 Essential oils The MIC values of O. asteriscoides essential oils individually and in combination with A. afra are given in Table 4.2. Even though the MIC values of A. afra essential oil have been discussed and evaluated in Chapter 3, Section 3.2.2.1, they have been shown in Table 4.2 for the sake of completeness. MIC values for O. asteriscoides essential oil ranged from as low as 1.0 mg/ml to 8.0 mg/ml. Noteworthy activity was seen against C. neoformans (MIC value = 2.0 mg/ml), S. pyogenes (MIC value = 1.7 mg/ml) and against M. smegmatis (MIC value = 1.3 mg/ml). The best MIC value of 1.0 mg/ml was obtained for O. asteriscoides against S. pneumoniae. 254 nm A White light C 365 nm B Derivatized D Aa Oa Aa + Oa Aa Oa Aa + Oa Aa Oa Aa + Oa Aa Oa Aa + Oa 90 Table 4.2 MIC (mg/ml) and ?FIC values of A. afra and O. asteriscoides essential oils alone and in combination. *Values given in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Viljoen et al. (2003) investigated the antimicrobial activity of O. asteriscoides essential oil using the disk diffusion, MIC and time-kill studies. MIC results for E. faecalis showed no activity at the highest concentration tested and an MIC value of 8.0 mg/ml against C. neoformans was obtained. The antimicrobial activity of O. asteriscoides is said to be mostly due to the high percentage of 1,8-cineole, which has been shown to have a broad spectrum of antimicrobial activity (Pattnaik et al., 1997). Historically cineole rich essential oils have been used in the treatment of respiratory conditions (Silvestre et al., 1999; Viljoen et al., 2003). Camphor, which is also a major compound found in O. asteriscoides, is known to be an antiseptic and a decongestant (Bruneton, 1995). Viljoen et al. (2003) tested the antimicrobial activity of 1,8-cineole and camphor independently and in combination and compared it to O. Micro-organism MIC (mg/ml) ?FIC A. afra O. asteriscoides A. afra with O. asteriscoides A. afra with O. asteriscoides (1:1) Interpretation K. pneumoniae NCTC 9633 8.0 8.0 8.0 1.0 Additive M. catarrhalis ATCC 23246 8.7 8.0 8.0 1.0 Additive E. faecalis ATCC 29212 8.7 8.0 8.0 1.0 Additive C. neoformans ATCC 90112 3.8 2.0 2.0 0.8 Additive S. pneumoniae ATCC 49619 3.3 1.0 1.0 0.7 Additive S. pyogenes ATCC 8668 1.7 1.7 1.5 0.9 Additive S. agalactiae ATCC 55618 4.8 4.7 7.0 1.5 Indifferent M. smegmatis (clinical) 1.5 1.3 1.5 1.1 Indifferent 91 asteriscoides essential oil and found that the two compounds act synergistically to enhance the overall activity of the oil. O. asteriscoides essential oil was tested by van Vuuren and Viljoen (2006) using the MIC micro-titre plate method and time-kill studies. MIC values were determined against S. aureus, S. epidermidis, B. cereus, B. subtilis, E. coli, P. aeruginosa, E. faecalis, K. pneumoniae, C. albicans and C. neoformans. Comparable pathogens, E. faecalis, K. pneumoniae and C. neoformans gave MIC values of 7.0, 11.9 and 1.0 mg/ml respectively. These results are in alignment with those obtained in the present study. Time-kill studies revealed bactericidal activity against S. aureus after 24 hrs. Against K. pneumoniae cidal activity was noted within one hour of exposure; however, regrowth occurred after four hours. Cidal effects were noted against C. albicans with four hours of exposure to the essential oil. In the study by van Vuuren (2007), MIC assays were performed on O. asteriscoides essential oil against ten pathogens. Two of these i.e. E. faecalis and C. neoformans, can be compared to the results obtained in this study. MIC values obtained by van Vuuren (2007) were 8.0 mg/ml for both pathogens. The results of this study are comparable with that of van Vuuren (2007) for the pathogen E. faecalis. However, for C. neoformans, the MIC determined in this study was much lower (2.0 mg/ml) . A possible explanation could be the difference in the chemical composition of the samples an due to the fact that the localities for sample collection were different, thus producing a different MIC. In the combination of O. asteriscoides with A. afra, noteworthy antimicrobial activity was noted against the same pathogens as was for O. asteriscoides i.e. C. neoformans (2.0 mg/ml), S. pneumoniae (1.0 mg/ml), S. pyogenes (1.5 mg/ml) and M. smegmatis (1.5 mg/ml). The MIC values of the combination against K. pneumoniae, M. catarrhalis and E. faecalis remained constant for the essential oils independently and in combination. Thus, the ?FIC values showed additive interactions when A. afra was combined with O. asteriscoides against these pathogens (?FIC = 1). Other ?FIC values of note were against C. neoformans, S. pneumoniae and S. pyogenes with values of 0.8, 0.7 and 0.9 respectively. Indifferent interactions were noted against S. agalactiae and M. smegmatis. No antagonism was noted for the combination against any of the pathogens tested. 92 4.2.2.1.2 Dichloromethane: methanol extracts The O. asteriscoides dichloromethane: methanol extract was active against all the pathogens tested (Table 4.3) with at least some antimicrobial activity noted (MIC values between 0.3 - 4.0 mg/ml). Activities for S. pneumoniae and S. pyogenes showed noteworthy sensitivity both with MIC values of 0.4 mg/ml, indicating O. asteriscoides is a strong inhibitor against these pathogens. The best activity was noted against C. neoformans where an MIC value of 0.3 mg/ml was obtained. Table 4.3 MIC and ?FIC values of A. afra and O. asteriscoides dichloromethane: methanol extracts alone and in combination. *Values given in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. For the combination with A. afra, some antimicrobial activity was noted against K. pneumoniae, M. catarrhalis, E. faecalis and S. agalactiae (MIC values between 1.0 - 6.0 mg/ml). Noteworthy activity was seen against C. neoformans, S. pneumoniae, S. pyogenes and M. smegmatis. A. afra with O. asteriscoides in combination were strong inhibitors against these four pathogens with MIC values ranging at 0.1 - 0.5 mg/ml. Micro-organism MIC (mg/ml) ?FIC A. afra O. asteriscoides A. afra with O. asteriscoides A. afra with O. asteriscoides (1:1) Interpretation K. pneumoniae NCTC 9633 6.3 4.0 6.0 1.2 Indifferent M. catarrhalis ATCC 23246 4.7 4.0 4.0 0.9 Additive E. faecalis ATCC 29212 6.3 2.0 4.0 1.3 Indifferent C. neoformans ATCC 90112 1.2 0.3 0.3 0.7 Additive S. pneumoniae ATCC 49619 0.5 0.4 0.1 0.3 Synergy S. pyogenes ATCC 8668 1.1 0.4 0.1 0.2 Synergy S. agalactiae ATCC 55618 3.0 1.3 1.0 0.6 Additive M. smegmatis (clinical) 1.7 1.4 0.5 0.3 Synergy 93 ?FIC values then calculated showed synergistic interactions with the combination against S. pneumoniae, S. pyogenes and M. smegmatis with values of 0.3, 0.2 and 0.3 respectively. Additive interactions were observed against M. catarrhalis, C. neoformans and S. agalactiae. Thus against all the pathogens tested for this combination, either synergy or additivity was noted with the exception of K. pneumoniae and E. faecalis which were indifferent. The dichloromethane: methanol extracts of O. asteriscoides have not to this date been tested for their antimicrobial activity. This study demonstrates for the first time the antimicrobial activity, using MIC methodology, of the dichloromethane: methanol extracts of O. asteriscoides independently and in combination. It is surprising that no studies have been done on the extracts as the traditional method of administration of O. asteriscoides is as a tincture. It is thus important that the alcohol extracts be tested for their activity in treating respiratory tract diseases. 4.2.2.1.3 Aqueous extracts The antimicrobial analysis of the aqueous extracts (Table 4.4) of O. asteriscoides yielded MIC values between 0.5 - 9.0 mg/ml against the pathogens tested. The lowest MIC value was against C. neoformans, with a value of 0.5 mg/ml. Moderate inhibition was also noted for O. asteriscoides against S. pneumoniae and S. agalactiae with values of 1.5 mg/ml. Against S. pyogenes the MIC value obtained was 1.8 mg/ml. For the combination of A. afra with O. asteriscoides some antimicrobial activity was obtained against S. pneumoniae and S. agalactiae with MIC values of 2.0 mg/ml, and 4.0 mg/ml against M. smegmatis. Moderate antimicrobial activity was recorded for S. pyogenes with an MIC value of 1.5 mg/ml. Strong noteworthy inhibitory activity and the lowest MIC value (0.5 mg/ml) was obtained against C. neoformans. ?FIC values obtained for the combination of A. afra with O. asteriscoides aqueous extracts showed additive interactions against K. pneumoniae, M. catarrhalis, E. faecalis, C. neoformans, S. pneumoniae, S. pyogenes and M. smegmatis. It is interesting to note none of the interactions of A. afra with O. asteriscoides yielded antagonistic effects. 94 Table 4.4 MIC and ?FIC values of A. afra and O. asteriscoides aqueous extracts alone and in combination. *Values given in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Scott et al. (2004) performed disc diffusion assays on the aqueous extracts of O. asteriscoides. Pathogens tested included S. aureus, P. aeruginosa, C. albicans and M. smegmatis. In their study, no activity of the aqueous infusion was noted against any of the pathogens tested. 4.2.2.2 Isobologram interpretation The isobologram of the combination of A. afra with O. asteriscoides essential oils, dichloromethane: methanol extracts and aqueous extracts against M. catarrhalis is shown in Figure 4.4. The essential oils show additive interactions for all the ratios tested. The dichloromethane: methanol extracts showed additive interactions for most of the ratios tested with two points as exceptions. These two points are synergistic and they are the ratios 1:9 (Figure 4.4, a) and 2:8 (Figure 4.4, b). Micro-organism MIC (mg/ml) ?FIC A. afra O. asteriscoides A. afra with O. asteriscoides A. afra with O. asteriscoides (1:1) Interpretation K. pneumoniae NCTC 9633 12.0 8.0 8.0 0.8 Additive M. catarrhalis ATCC 23246 8.0 8.0 8.0 1.0 Additive E. faecalis ATCC 29212 7.0 9.0 8.0 1.0 Additive C. neoformans ATCC 90112 4.0 0.5 0.5 0.6 Additive S. pneumoniae ATCC 49619 8.0 1.5 2.0 0.8 Additive S. pyogenes ATCC 8668 5.3 1.8 1.5 0.6 Additive S. agalactiae ATCC 55618 1.7 1.5 2.0 1.3 Indifferent M. smegmatis (clinical) 8.0 4.0 4.0 0.8 Additive 95 Figure 4.4 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against M. catarrhalis (a ? ratio 1:9; b ? ratio 2:8); ? 1:1combination. This synergy displayed between A. afra with O. asteriscoides takes place when the ratio of O. asteriscoides is in the majority. The aqueous extracts showed additive pharmacological interactions for five ratios tested. Four ratios (4:6, 3:7, 2:8 and 1:9) wherein O. asteriscoides dominates in the combination were synergistic. The 1:1 combination of the essential oils, dichloromethane: methanol extracts and the aqueous extracts are shown and depicted in Figure 4.4. The ?FIC values obtained for the 1:1 combination of the essential oils (Table 4.2), the dichloromethane: methanol extracts (Table 4.3) and the aqueous extracts (Table 4.4) against M. catarrhalis correlates with those results obtained in the isobolograms. The interactions of the combination of A. afra with O. asteriscoides against K. pneumoniae are graphically represented in Figure 4.5. The essential oils demonstrate additive interactions for all the ratios tested. The dichloromethane: methanol extracts show mainly indifferent interactions for six of the nine ratios tested. Three of the ratios i.e. 4:6, 3:7 and 1:9 were additive. Thus, when O. asteriscoides is in the majority in the combination, additive interactions are found. The aqueous extracts show additive interactions with all the ratios 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 M. catarrhalis ATCC 23246 MIC Aa in combination/MIC Aa independently M IC O a in co m bi na ti on /M IC O a in de pe nd en tl y a b 96 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 K. pneumoniae NCTC 9633 MIC Aa in combination/MIC Aa independently M IC O a in co m bi na ti on /M IC O a in d ep en d en tl y c tested except one, which was synergistic. The ratio showing synergy is depicted in Figure 4.5, (c) is when A. afra is in the majority at a ratio of 8:2. Figure 4.5 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against K. pneumoniae (c ? ratio 8:2); ?1:1 combination. The ?FIC values of the essential oils (Table 4.2), the dichloromethane: methanol extracts (Table 4.3) as well as the aqueous extract (Table 4.4) of the 1:1 combination against K. pneumoniae correlates well with the isobologram (Figure 4.5). The essential oils of the combination of A. afra with O. asteriscoides against E. faecalis are shown in Figure 4.6. All ratios for the combination showed additive interactions. The dichloromethane: methanol extracts showed additive interactions for most ratios tested. Two ratios tested were indifferent. These were at ratios 6:4 (where A. afra is dominant) and the 1:1 combination. The aqueous extracts showed additive interactions for all ratios except one (ratio 9:1) where A. afra was the dominant plant (Figure 4.6, d). This is interesting to note, as this information is not detected in the ?FIC determination method wherein only 1:1 combinations are interpreted. ?FIC data (Table 4.2, 4.3 and 4.4) and isobologram (Figure 4.6, 4.7 and 4.8) correlation was noted for the essential oils, dichloromethane: methanol and the aqueous extracts of the combination against E. faecalis. 97 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 MIC Aa in combination/MIC Aa independently M IC O a in co m bi na ti on /M IC O a in de pe nd en tl y E. faecalis ATCC 29212 d Figure 4.6 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against E. faecalis (d ? ratio 9:1); ? 1:1 combination. The interaction of A. afra and O. asteriscoides against C. neoformans is depicted in Figure 4.7. Results show the essential oil of the combination are additive for all the ratios tested except one which was synergistic (?FIC = 0.5). This was the ratio 8:2 (Figure 4.7, e), where A. afra essential oil is the dominant oil in the combination. The dichloromethane: methanol extracts displayed a mix of additive and indifferent interactions. The ratios in which indifference was noted were at 4:6, 3:7, 2:8 and 1:9. These are all ratios in which O. asteriscoides is in the majority. It is interesting to note that when A. afra and O. asteriscoides dichloromethane: methanol extracts are combined in equal parts, the interaction is additive. However, when the ratio of O. asteriscoides increases, the combination becomes indifferent. This is important to note as the traditional method of administration is a tincture. When traditional healers are mixing the combination together and if O. asteriscoides becomes more than A. afra, the administration of this combination could become less effective when treating C. neoformans related infections. It is reported that O. asteriscoides contains sesquiterpene lactones (Scott and Springfield, 2004), which may contribute to the possible toxicity. Thus, the possible harm is not due to any antagonism of the combination, but is from the possible toxicity and allergic 98 reactions of the sesquiterpene lactones present in O. asteriscoides (Scott and Springfield, 2004). Figure 4.7 Isobologram of the combination of A. afra and O. asteriscoides essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against C. neoformans (e ? ratio 8:2); ? 1:1 combination. When the aqueous extracts of A. afra and O. asteriscoides were combined against C. neoformans, additive and synergistic interactions were noted. Ratios 9:1, 8:2, 6:4, 4:6, 3:7, 2:8 and 1:9 were the synergistic combinations. It is surprising that the aqueous extracts have produced such good synergy and the dichloromethane: methanol extracts have resulted in indifferent activity. The ?FIC values obtained and the isobologram were congruent for the combination of the essential oils, dichloromethane: methanol extracts and the aqueous extracts against C. neoformans. Typically, the alcohol extracts produce better antimicrobial activity than the aqueous extracts. However, the combination is also administered as an infusion (Scott and Springfield, 2004) and although previous studies (Scott et al., 2004) have found no activity for the aqueous extracts, the results obtained in this study serves to validate the use of the combination of A. afra with O. asteriscoides in the form if an infusion. 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 C. neoformans ATCC 90112 MIC Aa in combination/MIC Aa independently M IC O a in co m bi na ti on /M IC O a in de pe nd en tl y e 99 4.3 Conclusions ? The major compounds present in the essential oil of O. asteriscoides were 1,8-cineole (59.0%), camphor (8.2%) and ?-terpineol (7.2%) making up 74.4% of the total composition of the oil (91.6%). ? The TLC studies of O. asteriscoides (essential oils, dichloromethane: methanol and the aqueous extracts) showed visible bands under different wavelengths of light and gave a rather characteristic fingerprint of the plants used in this assay. ? The best antimicrobial activity for the essential oil of O. asteriscoides was obtained against S. pneumoniae with a mean MIC value of 1.0 mg/ml. For the dichloromethane: methanol extracts, noteworthy inhibitory activity with MIC values of 0.33 mg/ml was obtained against C. neoformans, followed by S. pneumoniae and S. pyogenes (MIC values = 0.4 mg/ml. The best activity for the aqueous extract of O. asteriscoides was obtained against C. neoformans (MIC value of 0.5 mg/ml). ? The most prominent antimicrobial activity of the combination of A. afra with O. asteriscoides essential oils was obtained against S. pneumoniae (MIC value of 1.0 mg/ml) For the dichloromethane: methanol extracts, the most interesting and noteworthy activity was obtained against S. pneumoniae and S. agalactiae with MIC values of 0.1 mg/ml. The combination of the aqueous extracts gave the best noteworthy activity against C. neoformans (MIC value of 0.5 mg/ml). ? In the combination of the essential oils of A. afra and O. asteriscoides, ?FIC results revealed indifferent and additive pharmacological interaction. The dichloromethane: methanol extracts ?FIC results showed synergistic interactions against S. pneumoniae, S. pyogenes and M. smegmatis. The aqueous extracts gave various ?FIC results with additivity and indifference. ? Isobologram results of the combination show mainly additive interactions with the essential oils, with one point wherein eight parts of A. afra and two parts O. asteriscoides were combined (Figure 4.7, e) was synergistic. ? Synergistic interactions were found when tested against M. catarrhalis with the dichloromethane: methanol extracts at ratios 1:9 and 2:8 (Figure 4.4, a and b). The aqueous extracts showed synergy at ratios 4:6, 3:7, 2:8 and 1:9 against M. catarrhalis. ? Against K. pneumoniae, synergy was noted for one ratio i.e. at eight parts A. afra and two parts O. asteriscoides (Figure 4.5, c) of the combination of the aqueous extracts. 100 ? The aqueous extracts showed synergy against E. faecalis at nine parts A. afra and one part O. asteriscoides (Figure 4.6, d). ? The best activity was obtained for the combination of A. afra with O. asteriscoides aqueous extracts against C. neoformans, where seven of the nine ratios tested were synergistic (Figure 4.7). ? No antagonism was noted for the combination against any of the pathogens tested. ? The outcomes of this chapter and the testing of the combination of A. afra with O. asteriscoides dichloromethane: methanol extracts provides in vitro evidence to support the use of a tincture in traditional African healing for the treatment of respiratory tract infections. Moreover, the aqueous extracts have also provided in vitro evidence of efficaciousness in the traditional use of the combination of A. afra with O. asteriscoides. 101 Chapter 5 The antimicrobial efficacy of Artemisia afra Jacq. Ex Willd and Agathosma betulina (P.J. Berguis) Pillans in combination. 5.1 Introduction A. betulina is said to be one of the best-known medicinal plants of Southern Africa, which is used both locally and internationally for the treatment of various diseases (Moolla et al., 2007; Moolla and Viljoen, 2008). Traditionally, A. betulina is used in combination with A. afra and taken for colds and influenza or as a general tonic (Scott and Springfield, 2004). Traditionally 1% (15 mg/ml) of buchu extract is added in combination with A. afra (1.5% (10 mg/ml) of an extract) to a white sweet wine to make ?The South African herbal wine? (Watt and Breyer-Brandwijk, 1962). Buchu and African wormwood have been studies extensively independently; however, the combination of the plants has never been evaluated scientifically. These two plants are widely used individually, generally well-accepted and respected plant medicines. Thus the reason of adding this combination to this study. 5.2 Results and discussion 5.2.1 Chromatographic techniques 5.2.1.1Essential oil composition of A. betulina The essential oil composition of buchu revealed a total number of 30 compounds making up 97.5% of the total composition (Table 5.1). The major compounds were identified as limonene (18.9%), menthone (10.0%), isomenthone (31.4%), pseudodiosphenol (7.5%), and diosphenol (8.1%). Previous studies on the chemical composition of buchu oil are discussed in Appendix A1. In brief, Kaiser et al. (1975) identified the main constituent as isomethone (50-60%) of the essential oil of buchu. Viljoen et al. (2006a) identified the major compound in commercial buchu oil purchased from Afriplex?, South Africa as menthone (29.2%). Limonene was also present (23.7%). The main constituent found in A. betulina essential oil in this study, was isomethone at 31.4%. Smaller quantities of menthone (10.0%) were found. Pulegone, a highly toxic compound noted by Viljoen et al. (2006a) at 8.4%, was also found in this study but at lower concentrations (4.1%). 102 Table 5.1 Essential oil composition of A. betulina. *RRI: Relative retention indices calculated against n-alkanes. % calculated from TIC data. From the literature, there are many other reports on the chemical composition of the essential oil of A. betulina (Gentry, 1961; Watt and Breyer-Brandwijk, 1962; Kaiser et al., 1975; Simpson, 1998; Lis-Balchin et al., 2001; van Wyk and Wink, 2004; Moolla, 2006; Viljoen et al., 2006a; Moolla et al., 2007; Moolla and Viljoen, 2008; van Wyk et al., 2009;). However, interest is only recently being directed towards the study of the non-volatile fraction of this RRI* Compounds Area (%) 1016 ?-Pinene 0.8 1104 ?-Pinene. 0.3 1117 Sabinene 0.8 1159 Myrcene 1.5 1192 ?-Terpinene 0.1 1193 Limonene 18.9 1202 1,8-Cineole 1.7 1242 ?-Terpinene tr 1250 (E)-?-Ocimene 0.5 1270 p-Cymene 0.5 1475 Menthone 10.0 1468 Fenchyl acetate 0.1 1496 Isomenthone 31.4 1546 Linalool 0.5 1572 Neoisopulegol 0.1 1577 cis Isopulegone 3.3 1587 trans Isopulegone 2.8 1602 Terpinen-4-ol 0.4 1610 Dihydrocarvone 0.2 1653 Pulegone 4.1 1692 Myrtenyl acetate 0.1 1701 ?-Terpineol 0.1 1715 Germacrene D 0.1 1739 Piperitone 0.2 1752 Pseudodiosphenol 7.5 1825 Diosphenol 8.1 1873 trans-8-Mercapto-p-mentha- 3-one 0.4 1905 cis-8-Mercapto-p-mentha-3- one 2.3 2082 Methyl eugenol 0.5 2188 Eugenol 0.2 Total 97.5 103 plant (Moolla et al., 2007; Vermaak et al., 2009). The importance of studying a plant in its entirety cannot be emphasized enough as the possibility of interactions of volatile and non- volatile compounds within a plant need to be considered as well as the individual contributions of each component (van Vuuren and Viljoen, 2009). 5.2.1.2 Thin layer chromatography Thin layer chromatography analysis of the essential oils and the dichloromethane: methanol extracts of A. afra (discussed in Chapter 3), A. betulina and the combination of the two, revealed the presence of fluorescent compounds, which were visible under UV light of 254 nm as well as 365 nm. The aqueous extracts did not show good separation of the compounds and thus were not included. Figure 5.1 shows the chromatograms derived for the essential oils. A black fluorescent compound in A. betulina, at Rf = 0.5 was seen at 254 nm as well as at 365 nm (lighter in colour). Another major compound was detected in A. betulina at 365 nm at Rf = 0.6. When derivatized these compounds and various others were noted. Figure 5.1 TLC fingerprints of the essential oils of A. afra and A. betulina alone and in combination at 254 nm, 365 nm and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Ab ? A. betulina; Aa + Ab ? A. afra with A. betulina. The dichloromethane: methanol extracts (Figure 5.2) showed the presence of many compounds in A. betulina and in the combination with A. afra. At 254 nm major compounds were seen at Rf = 0.65 and Rf = 0.8. At 365 nm a compound showing up red was detected with an Rf = 0.65 as well. Under white light, the same compound is detected with the same Rf value. When derivatized the compound is not as visible but is mixed in with other visualized compounds. 254 nm A 365nm B Derivatized C Aa Ab Aa +Ab Aa Ab Aa + Ab Aa Ab Aa + Ab 104 Figure 5.2 TLC fingerprints of the dichloromethane: methanol extracts of A. afra and A. betulina alone and in combination at 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Ab ? A. betulina; Aa + Ab ? A. afra with A. betulina. 5.2.2 Antimicrobial analysis 5.2.2.1 MIC assays and FIC determination 5.2.2.1.1 Essential oils The MIC values and of the essential oil of A. betulina individually and in combination with A. afra are given in Table 5.2. The MIC values of A. betulina essential oils ranged between 1.0-16.0 mg/ml depending on the pathogen tested. The lowest MIC value was noted against S. pyogenes with noteworthy activity (MIC value of 1.0 mg/ml). Other noteworthy activity obtained was against S. pneumoniae and M. smegmatis (MIC values of 2.0 mg/ml). Some antimicrobial activity was obtained against C. neoformans, giving a MIC value of 6.0 mg/ml. Very low antibacterial activity was obtained against K. pneumoniae, M. catarrhalis, E. faecalis and S. agalactiae (MIC values ?8.0 mg/ml). The antimicrobial activity of the essential oil of buchu was previously studied using the agar diffusion method (Lis-Balchin et al., 2001). The pathogens tested included E. coli, Enterococcus hirae, P. aeruginosa, Saccharamyces cerevisiae and S. aureus. Very poor to no antimicrobial activity was obtained against the pathogens. In another study, the antimicrobial activity of the essential oil of A. betulina was tested using the MIC micro-titre plate method (Moolla, 2006; Viljoen et al., 2006a). Essential oils tested were from Landmeterskop, Middelberg by Moolla, (2006) and a commercial product was 254 nm A 365 nm B White light C Derivatized D Aa Ab Aa +Ab Aa Ab Aa + Ab Aa Ab Aa +Ab Aa Ab Aa+ Ab 105 tested by Viljoen et al., (2006a). Some activity was noted against the pathogens tested i.e. B. cereus, C. albicans, K. pneumoniae and S. aureus with MIC values from 4.0-32.0 mg/ml. Results obtained against K. pneumoniae (MIC value = 8.0 mg/ml) in the current study were congruent with those mentioned in the review by Moolla and Viljoen, (2008). Table 5.2 MIC and ?FIC values of essential oils of A. afra and A. betulina, alone and in combination. *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. For the combination of A. afra with A. betulina, the lowest MIC value with noteworthy activity (2.0 mg/ml) obtained against the pathogens S. pneumoniae, S. pyogenes and M. smegmatis. Against C. neoformans and S. agalactiae some antimicrobial activity was noted (MIC values of 4.0 mg/ml). Very low activity was seen against K. pneumoniae, M. catarrhalis and E. faecalis with MIC values of 8.0 mg/ml. Micro-organism MIC (mg/ml) ?FIC A. afra A. betulina A. afra with A. betulina A. afra with A. betulina (1:1) Interpretation K. pneumoniae NCTC 9633 8.0 8.0 8.0 1.0 Additive M. catarrhalis ATCC 23246 8.7 16.0 8.0 0.7 Additive E. faecalis ATCC 29212 8.7 8.0 8.0 1.0 Additive C. neoformans ATCC 90112 3.8 6.0 4.0 0.9 Additive S. pneumoniae ATCC 49619 3.3 2.0 2.0 0.8 Additive S. pyogenes ATCC 8668 1.7 1.0 2.0 1.6 Indifferent S. agalactiae ATCC 55618 4.8 8.0 4.0 0.7 Additive M. smegmatis (clinical) 1.5 2.0 2.0 1.2 Indifferent 106 ?FIC values obtained against K. pneumoniae, M. catarrhalis, E. faecalis, C. neoformans S. pneumoniae and S. agalactiae displayed additive pharmacological interactions. Indifferent interactions were noted against S. pyogenes and M. smegmatis. Importantly, however, the combination of A. afra with A. betulina essential oil did not illustrate any antagonism. 5.2.2.1.2 Dichloromethane: methanol extracts The dichloromethane: methanol extracts of A. betulina showed some antimicrobial activity (Table 5.3) against C. neoformans, S. pneumoniae, S. pyogenes, S. agalactiae and M. smegmatis with MIC values ranging from 1.4-4.0 mg/ml. Moderate inhibition was noted against S. pneumoniae (MIC value of 1.4 mg/ml), S. pyogenes (1.5 mg/ml) and M. smegmatis (1.5 mg/ml). Very poor activity (MIC value of 16.0 mg/ml) was obtained against M. catarrhalis while no activity was seen against K. pneumoniae and E. faecalis at the highest concentration tested. A. afra independently showed some antimicrobial activity as noted in Chapter 3. Moolla et al. (2007) explored the biological activity of the non-volatile fraction of Agathosma species. The dichloromethane: methanol extracts of A. betulina from Landmeterskop, Middelberg, were tested using the MIC micro-titre plate method against B. cereus, S. aureus, K. pneumoniae and C. albicans. The MIC values obtained (2.0-4.0 mg/ml) showed the presence of some activity. The MIC value obtained against K. pneumoniae was 4.0 mg/ml. Sandasi (2008) investigated the antimicrobial activity of the essential oils, dichloromethane: methanol and the methanol extracts of A. betulina. MIC micro-titre plate assays were conducted on seven micro-organisms i.e. L. monocytogenes, P. aeruginosa, C. albicans, E. coli, P. vulgaris S. aureus and E. faecalis. The greatest activity was seen with the dichloromethane: methanol extracts with MIC values from 3.0-6.0 mg/ml. these results correlate with the findings reported here. In the current study, the best result was obtained in the combination of A. afra with A. betulina against S. pneumoniae with a MIC value of 0.6 mg/ml. This demonstrates that the combination of A. afra with A. betulina is far superior to when used independently for the treatment of microbial infections. 107 Table 5.3 MIC and ?FIC values of dichloromethane: methanol extracts of A. afra and A. betulina, alone and in combination. *Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for A. betulina was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. The combination of A. afra with A. betulina showed noteworthy antimicrobial activity against S. pneumoniae with an MIC value of 0.6 mg/ml. Some antimicrobial activity was seen against M. catarrhalis, C. neoformans, S. pyogenes, S. agalactiae and M. smegmatis with moderate inhibition against C. neoformans, S. pyogenes and M. smegmatis (MIC values of 1.3-2.0 mg/ml). Poor activity was observed for K. pneumoniae and E. faecalis. A very interesting synergistic reaction to the combination against M. catarrhalis occurred wherein a ?FIC value of 0.3 was noted. ?FIC values could not be calculated for the interactions against K. pneumoniae and E. faecalis as no end point for A. betulina was obtained. However, a tentative interpretation of Micro-organism MIC (mg/ml) ?FIC A. afra A. betulina A. afra with A. betulina A. afra with A. betulina (1:1) Interpretation K. pneumoniae NCTC 9633 6.3 ?16.0 10.3 ND1 Additive/indifferent2 M. catarrhalis ATCC 23246 4.7 16.0 2.0 0.3 Synergy E. faecalis ATCC 29212 6.3 ?16.0 8.0 ND1 Additive/indifferent2 C. neoformans ATCC 90112 1.2 4.0 1.3 0.7 Additive S. pneumoniae ATCC 49619 0.5 1.4 0.6 0.8 Additive S. pyogenes ATCC 8668 1.1 1.5 1.3 1.0 Additive S. agalactiae ATCC 55618 3.0 2.5 2.0 0.7 Additive M. smegmatis (clinical) 1.7 1.5 1.3 0.8 Additive 108 the interaction is given. The MIC results of the combination compared to the individual plant extracts against K. pneumoniae and E. faecalis showed that the interaction is probably additive or indifferent. ?FIC values calculated against C. neoformans, S. pneumoniae, S. pyogenes, S. agalactiae and M. smegmatis gave additive interactions for the combination. The antimicrobial activity of the combination was mostly additive indicating a positive rationale for the use with A. afra for the treatment of respiratory related diseases. 5.2.2.1.3 Aqueous extracts The aqueous extracts (Table 5.4) of A. betulina were not active against any the pathogens at the highest concentration tested. Table 5.4 MIC and ?FIC values of aqueous extracts of A. afra and A. betulina, alone and in combination. *Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for A. betulina was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml) ?FIC A. afra A. betulina A. afra with A. betulina A. afra with A. betulina (1:1) Interpretation K. pneumoniae NCTC 9633 12.0 ?16.0 8.0 ND1 Additive/indifferent2 M. catarrhalis ATCC 23246 8.0 ?16.0 ?16.0 ND1 Indifferent/antagonistic2 E. faecalis ATCC 29212 8.0 ?16.0 8.0 ND1 Additive/indifferent2 C. neoformans ATCC 90112 4.0 ?16.0 5.0 ND1 Additive/indifferent2 S. pneumoniae ATCC 49619 8.0 ?16.0 ?16.0 ND1 Indifferent/antagonistic2 S. pyogenes ATCC 8668 5.3 ?16.0 ?16.0 ND1 Indifferent/antagonistic2 S. agalactiae ATCC 55618 1.7 ?16.0 ?16.0 ND1 Indifferent/antagonistic2 M. smegmatis (clinical) 8.0 ?16.0 ?16.0 ND1 Indifferent/antagonistic2 109 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 M. catarrhalis ATCC 23246 MIC Aa in combination/MIC Aa independently M IC A b in co m bi na ti on /M IC A b in de pe nd en tl y a b Some poor activity was noted for the combination of A. afra with A. betulina aqueous extracts against K. pneumoniae, E. faecalis and C. neoformans (MIC values between 5.0-8.0 mg/ml). Due to the lack of susceptibility of any of the selected pathogens to the extracts of A. betulina, ?FIC values could not be calculated. Tentative interpretations of the interactions based on the comparison of the MIC values were made. Additive/indifferent interactions were assumed for the combination against K. pneumoniae, E. faecalis and C. neoformans. Against M. catarrhalis, S. pneumoniae S. pyogenes, S. agalactiae and M. smegmatis, the interaction was interpreted as indifferent/antagonistic. 5.2.2.2 Isobologram interpretation The isobolograms of the interactions of A. afra with A. betulina essential oils against M. catarrhalis (Figure 5.3) showed additive interactions for most of the ratios tested. Two ratios showed indifference i.e. 8:2 and 7:3 (Figure 5.3, a) where A. afra is in the majority. The dichloromethane: methanol extracts showed activity wherein three ratios were synergistic, four were additive and two of them indifferent. The ratios showing indifference are 2:8 and 1:9, wherein A. betulina is in the majority. The synergistic interactions occur at the ratios 3:7, 4:6 and 1:1 (Figure 5.3, b). The ?FIC interpretation data correlates with the 1:1 interaction where a FIC value of 0.3 was noted (Table 5.3). Figure 5.3 Isobologram of the combination of A. afra and A. betulina essential oils ( ) and the dichloromethane: methanol extracts ( ) against M. catarrhalis (a ? ratios 8:2 and 7:3; b ? ratios 3:7, 4:6 and 1:1); ? 1:1 combination. 110 The isobologram of the combination of A. afra with A. betulina against the pathogen K. pneumoniae showed additive interactions with all the ratios of the essential oil combination tested (Figure 5.4). Figure 5.4 Isobologram of the combination of A. afra and A. betulina essential oils ( ) against K. pneumoniae; ? 1:1 combination. Figure 5.5 depicts the interactions of A. afra with A. betulina against E. faecalis. The essential oils show additive interactions for all the ratios tested with one point (9:1) showing indifference (Figure 5.5, c). Figure 5.5 Isobologram of the combination of A. afra and A. betulina essential oils ( ) against E. faecalis (c ? ratio 9:1); ? 1:1 combination. 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 K. pneumoniae NCTC 9633 MIC Aa in combination/MIC Aa independently M IC A b in co m bi na ti on /M IC A b in de pe nd en tl y 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 E. faecalis ATCC 29212 MIC Aa in combination/MIC Aa independently M IC A b in co m bi na ti on /M IC A b in de pe nd en tl y c 111 The pathogen C. neoformans, tested for antimicrobial susceptibility to various combinations of A. afra and A. betulina essential oils are illustrated in Figure 5.6. Figure 5.6 Isobologram of the combination of A. afra and A. betulina essential oils ( ) and dichloromethane: methanol extracts ( ) against C. neoformans (d ? ratio 7:3, e ? ratio 1:9); ? 1:1 combination. The combination proved to give additive interactions for all ratios tested. The dichloromethane: methanol extracts were additive in this combination for most of the ratios tested with the exception of two (7:3 and 1:9). These two points (Figure 5.6, d and e) proved to be indifferent in their interactions against C. neoformans. The interpretation of the interactions of the 1:1 combination of A. afra with A. betulina essential oils against M. catarrhalis, K. pneumoniae, E. faecalis and C. neoformans showed congruency between the ?FIC determination and the isobologram method. The combination was also congruent for the dichloromethane: methanol extracts against M. catarrhalis and C. neoformans. The dichloromethane: methanol extracts against K. pneumoniae and E. faecalis did not yield an end point MIC value for A. betulina, thus isobolograms could not be drawn. The MIC values of the combination of different ratios of the dichloromethane: methanol extracts of A. afra with A. betulina are shown in Figure 5.7. 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 C. neoformans ATCC 90112 MIC Aa in combination/MIC Aa independently M IC A b in co m bi na ti on /M IC A b in de pe nd en tl y d e 112 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10 M IC val u e (m g/ m l) Ratio (A. afra: A. betulina) K. pneumoniae E. faecalis Figure 5.7 MIC values of the different ratios of the combination of A. afra with A. betulina dichloromethane: methanol extracts against K. pneumoniae and E. faecalis. The graph depicts lower MIC values for ratios 9:1, 8:2 and 7:3 against K. pneumoniae. These MIC values (3.0 mg/ml) occur when A. afra is the dominant extract in the combination. These ratios can therefore be interpreted as being synergistic or additive. As the ratio of A. betulina increases, so does the MIC value. The MIC values drop again at ratios 2:8 and 1:9 when the ratio of A. betulina is higher. Thus at different ratios, the interaction between the combination varies. This can be interpreted as synergy or additivity at very specific and narrow ratios. The MIC values obtained for the nine ratios of the dichloromethane: methanol extracts of A. afra with A. betulina tested against E. faecalis, showed an increase in MIC value as the ratio of A. betulina increases. The graph serves to demonstrate the change in MIC value as the ratio of plant extracts change. The aqueous extracts of the combination of A. afra with A. betulina against M. catarrhalis, E. faecalis, K. pneumoniae and C. neoformans could not be plotted onto the isobologram, as A. betulina had no end point MIC value. The graph constructed using the MIC data for all the >16 113 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10 M IC val u e (m g/ m l) Ratio (A. afra: A. betulina) K. pneumoniae M. catarrhalis E. faecalis C. neoformans ratios tested are shown (Figure 5.8). The MIC values generally increases as the percentage of A. betulina increases in the ratio combination with A. afra. Figure 5.8 The MIC values of the different ratios of the combination of A. afra with A. betulina aqueous extracts against K. pneumoniae, M. catarrhalis, E. faecalis and C. neoformans. The results from this study have demonstrated that A. afra and A. betulina may have a better inhibitory effect depending on the dosage forms in which they are tested. This may have selective effects on the interactions of the combination and thus confirms that method of administration is an essential area for future studies in order to better understand the use of combinations in traditional medicine. Here, the essential oils showed the most additivity in the combination and therefore may validate the use of these plants in an inhaled form. The solvent extracts of the combination showed the only evidence of synergy against M. catarrhalis. This is an interesting aspect as the most common use of this combination is an herbal wine. >16 114 5.3 Conclusions ? The main constituents found in the essential oil (97.5% of the total composition) of A. betulina were limonene (18.9%), menthone (10.0%), isomenthone (31.4%), pseudodiosphenol (7.5%), and diosphenol (8.1%). ? TLC showed the presence of many compounds visible at 254 nm and 365 nm. After derivatization with vanillin-sulphuric acid reagent, many more compounds were made visible. ? The MIC studies revealed the best antimicrobial activity for the essential oil of A. betulina was against S. pyogenes with a MIC value of 1.0 mg/ml. The best and most noteworthy activity of the combination of the essential oil was against S. pneumoniae, S. pyogenes and M. smegmatis (MIC values of 2.0 mg/ml). ? The dichloromethane: methanol extracts of the combination of A. afra with A. betulina gave the best activity against S. pneumoniae with a MIC value of 0.6 mg/ml. ? A. betulina aqueous extracts were inactive at the concentration tested against all eight pathogens. The combination with A. afra showed some activity against K. pneumoniae, E. faecalis and C. neoformans. ? Synergy was noted for the combination of the dichloromethane: methanol extracts against M. catarrhalis with ?FIC value of 0.3. ? Synergy was noted for three ratios in the isobologram of the dichloromethane: methanol extracts against M. catarrhalis. ? The predominantly additive interactions of the essential oils and the synergy of the dichloromethane: methanol extracts against M. catarrhalis observed in the combination of A. afra with A. betulina give credibility and provides preliminary evidence to support the traditional uses in the treatment of respiratory tract infections. 115 Chapter 6 The antimicrobial efficacy of Artemisia afra Jacq. Ex Willd and Eucalyptus globulus Labill. In combination 6.1 Introduction Although E. globulus is not indigenous to South Africa, this medicinal plant is used extensively in African traditional herbal medicine. The combination of A. afra with E. globulus is one that is used frequently in the treatment of flu-like symptoms and chest-related complaints. The combination of the leaf of A. afra and the leaf of E. globulus has been said to be used either externally or internally as an influenza remedy (Smith, 1895; Watt and Breyer- Brandwijk, 1962). Additionally a decoction of the combination is used for the common cold or catarrh (Smith, 1895). The steam from a strong decoction of the leaves of the combination of these two plants is said to relieve immediate symptoms (Smith, 1895). Thus, it is important to include in the evaluation of A. afra plant combinations. It is not only commonly used by traditional healers but it also demonstrates the versatility of the traditional healers and their practices to incorporate a variety of medicinal plants into their treatment regimen. 6.2 Results and discussion 6.2.1 Chromatographic techniques 6.2.1.1 Essential oil composition of E. globulus Analysis of the essential oils constituents of E. globulus revealed 22 compounds (Table 6.1) by comparison of mass spectral data and retention times of authentic compounds. The main compound found was 1,8-cineole making up 63% of the total composition of the essential oil (96.4%). Other major compounds identified were ?-pinene (16.7%), limonene (3.6%) and ?- cymene (2.7%). The essential oil of E. globulus has been well characterized (Batista-Pereira et al., 2006; Mayaud et al., 2008; George et al., 2009; Tohidpour et al., 2010). Mayaud et al. (2008) performed GC-MS analysis on the essential oil of E. globulus supplied by Pranar?m 116 (International S.A., Ghislenghein, Belgium) and found the main constituents to be 1,8-cineole (80.4%), limonene (7.47%), ?, ? and ?-terpinene (3.7%) and ?-cymene (2.5%). Table 6.1 Essential oil constituents of E. globulus. *RRI: Relative retention indices calculated against n-alkanes. % calculated from TIC data. George et al. (2009) studied the chemical variation of E. globulus from China. The main constituents found were 1,8-cineole (81.9%), D-limonene (7.6%), ?-pinene (3.3%) and benzene (3.4%). Previously Cimanga et al. (2002) studied the essential oil composition of E. globulus and found less of the 1,8-cineole (44.3%) than George et al. (2009). It is very common to find variation in the chemical composition of an essential oil between different varieties or within a plant (Moreno et al., 2007; George et al., 2009). Tohidpour et al. (2010) found the major constituents of the essential oil of E. globulus from Iran to be eucalyptol (47.2%), as the major constituent and most characteristic component of E. globulus, (+) spathulenol (18.1%) and ?-pinene (9.6%). The percentage of 1,8-cineole found in this study (Table 6.1) was not as much as that of Mayaud et al. (2008) and George et al. (2009), but neither was it as low as that found by Tohidpour et al. (2010). The results of the chemical RRI* Compounds Area (%) 1016 ?-Pinene 16.7 1104 ?-Pinene 0.3 1193 Limonene 3.6 1202 1,8-Cineole 63.0 1270 p-Cymene 2.7 1436 p-Cymenene 0.1 1441 trans-Linalool oxide 0.1 1471 cis-Linalool oxide 0.1 1521 Camphor 0.1 1546 Linalool 0.3 1573 Pinocarvone 0.6 1583 Fenchol 0.1 1590 Calarene 0.1 1602 Terpinen-4-ol 0.6 1604 Aromadendrene 1.2 1647 allo Aromadendrene 0.2 1662 trans Pinocarveol 1.1 1701 ?-Terpineol 1.6 1709 Borneol 0.2 1876 cis Carveol 0.2 2031 epi-Globulol 0.7 2094 Globulol + cubenol 2.8 Total 96.4 117 composition of E. globulus essential oil found in the current study are consistent with the percentage of 1,8-cineole found by George et al. (2009) where the main compound found was 1,8-cineole at 63.0% of the total composition of the essential oil. 6.2.1.2 Thin layer chromatography The presence of major compounds is detected using TLC (Ahmad and Beg, 2001). Compounds in E. globulus that are responsible for activity are alkaloids, phenols and tannins, as described by Ahmad and Beg (2001). The essential oil (Figure 6.1) of E. globulus showed two compounds at 254 nm. These were at Rf values of 0.45 and 0.5. No fluorescent compounds were visible under 365 nm. After derivatization, many compounds became visible. Compound at Rf values of 0.9, 0.25, 0.35 and 0.4 were some that were seen. Once again, the TLC chromatograms showed the complexity of the essential oils alone and the increased complexity in combination. Figure 6.1 TLC of the essential oils of A. afra and E. globulus individually and in combination at 254 nm, 365 nm and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Eg ? E. globulus; Aa + Eg ? A. afra with E. globulus. The TLC chromatograms of the dichloromethane: methanol extracts are depicted in Figure 6.2 Good separation is seen for E. globulus individually and in combination with A. afra. At 254 nm, many compounds between Rf value 0.35 and 0.55 as well as between Rf 0.8 and 0.9 were seen. Two highly fluorescent red compounds are seen in E. globulus at 365 nm at Rf = 0.65 and 0.9 respectively. 254 nm A 365 nm B Derivatized C Aa Eg Aa + Eg Aa Eg Aa + Eg Aa Eg Aa + Eg 118 Figure 6.2 TLC of the dichloromethane: methanol extracts of A. afra and E. globulus individually and in combination at 254 nm, 365 nm, visualized under white light and visualized with vanillin-sulphuric acid reagent. Aa ? A. afra; Eg ? E.globulus; Aa + Eg ? A. afra with E. globulus. The aqueous extracts of E. globulus and its combination with A. afra are shown in Figure 6.3. At 254 nm, few compounds are visible. Two compounds in E. globulus at Rf values of 0.55 and 0.6 are seen at 365 nm, compounds were not visible. Under white light, the separation of compounds showed up mostly yellow. Figure 6.3 TLC of the aqueous extracts of A. afra and E. globulus individually and in combination at 254 nm, 365 nm, visualized under white light and visualized with vanillin- sulphuric acid reagent. Aa ? A. afra; Eg ? E. globulus; Aa + Eg ? A. afra with E. globulus. 254 nm A 365 nm B Derivatized C Aa Eg Aa + Eg Aa Eg Aa + Eg Aa Eg Aa + Eg Aa Eg Aa + Eg White light C 254 nm A 365 nm B White light C Derivatized D Aa Eg Aa + Eg Aa Eg Aa + Eg Aa Eg Aa + Eg Aa Eg Aa + Eg 119 Compounds were much more visible after derivatization for E. globulus and the combination (Figure 6.3). Blue compounds were seen for E. globulus and in the combination at Rf values of 0.39, 0.41 and 0.76. 6.2.2 Antimicrobial analysis 6.2.2.1 MIC assays and FIC determination 6.2.2.1.1 Essential oils Antimicrobial studies of E. globulus essential oils (Table 6.2) showed diverse results. E. globulus against K. pneumoniae, M. catarrhalis and E. faecalis showed very poor antimicrobial activity with MIC values of 8.0 mg/ml. Some antimicrobial activity was obtained against S. pneumoniae (MIC value = 2.7 mg/ml) and S. agalactiae (5.3 mg/ml). Noteworthy activity for the essential oil E. globulus was obtained against S. pyogenes (MIC value = 2.0 mg/ml) and M. smegmatis (MIC value = 2.0 mg/ml). The best activity was noted against C. neoformans yielding an MIC value of 0.6 mg/ml, Cimanga et al. (2002) tested the antimicrobial activity of E. globulus essential oil using the disc diffusion method. The pathogens included E. coli, B. subtilis, K. pneumoniae, P. aeruginosa, S. aureus and S. flexneri. The activity against two K. pneumoniae strains were interpreted as very active and against P. aeruginosa the oil was moderately active. The antimicrobial properties of the essential oil of E. globulus against S. aureus and E. coli were confirmed using the aromatogramme, microastmosphere and germs in suspension methods (Ghalem and Mohamed, 2008a). In another study on the antimicrobial activity of the essential oil of E. globulus, the agar dilution method was used to calculate mean MIC percentages (Mayaud et al., 2008). Activity was assessed on a number of micro-organisms including K. pneumoniae (mean MIC = 10.0%) and E. faecalis (mean MIC = 5.83 ?SD of 1.86%). Twelve Streptococci spp were also evaluated for their potential susceptibility (mean MIC = 2.78?SD of 2.08%). Good reproducibility and results were found in terms of MIC's. The antimicrobial activity of an essential oil is linked to the chemical composition of the oil i.e. the presence of alcohols, phenols, terpenes and ketones (Iserin, 1997; Inouye et al., 2001; Cimanga et al., 2002; Ghalem and Mohamed, 2008a, b). 120 Table 6.2 MIC and ?FIC values the essential oils of A. afra and E. globulus alone and in combination. Micro-organism MIC (mg/ml) ?FIC A. afra E. globulus A. afra with E. globulus A. afra with E. globulus (1:1) Interpretation K. pneumoniae NCTC 9633 8.0 8.0 8.0 1.0 Additive M. catarrhalis ATCC 23246 8.7 8.0 8.0 1.0 Additive E. faecalis ATCC 29212 8.7 8.0 8.0 1.0 Additive C. neoformans ATCC 90112 3.8 0.6 0.8 0.8 Additive S. pneumoniae ATCC 49619 3.3 2.7 3.0 1.0 Additive S. pyogenes ATCC 8668 1.7 2.0 1.5 0.8 Additive S. agalactiae ATCC 55618 4.8 5.3 4.0 0.8 Additive M. smegmatis (clinical) 1.5 2.0 1.3 0.7 Additive *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Recently Tohidpour et al. (2010) tested the essential oil of E. globulus using the disk diffusion and the agar diffusion methods. Zones of inhibition and MIC values were calculated for B. cereus, E. coli, K. pneumoniae, Methicillin resistant S. aureus (MRSA) and S. aureus. E. coli was the most sensitive pathogen tested with an inhibition zone of 8 mm and a MIC value of 51.36 ?g/ml. K. pneumoniae yielded an inhibition zone of 8 mm and the MIC value obtained was 68.48 ?g/ml. Some variation against K. pneumoniae is evident between this current study and that undertaken by Tohidpour et al. (2010). This is possibly due to the different methods of evaluation of antimicrobial activity that were used. In the combination of A. afra and E. globulus essential oils, the same trend of antimicrobial activity as that of E. globulus alone was noticed. Very poor activity was seen against K. pneumoniae, M. catarrhalis and E. faecalis (MIC values of 8.0 mg/ml). Some antimicrobial 121 activity was seen against S. pneumoniae (MIC value = 3.0 mg/ml) and S. agalactiae (4.0 mg/ml). Noteworthy activity was obtained against C. neoformans yielding the best MIC value of the combination of 0.8 mg/ml, S. pyogenes (MIC value = 1.5 mg/ml) and M. smegmatis (MIC value = 1.3 mg/ml). The ?FIC values calculated (Table 6.2) for the combination of A. afra with E. globulus essential oil against all the pathogens tested, yielded additive pharmacological interactions. 6.2.2.1.2 Dichloromethane: methanol extracts The dichloromethane: methanol extract of E. globulus against all the pathogens tested, showed antimicrobial activity with MIC values between 0.01 - 3.0 mg/ml (Table 6.3). Some activity was noted against K. pneumoniae (MIC value = 3.0 mg/ml). Moderate inhibitory antimicrobial activity was noted against S. agalactiae and M. smegmatis with MIC values of 1.0 and 1.5 mg/ml respectively. Against M. catarrhalis and E. faecalis, C. neoformans, S. pneumoniae and S. pyogenes noteworthy activity was obtained for E. globulus. M. catarrhalis and E. faecalis exhibited very strong inhibitory activity with MIC values of 0.01 mg/ml and 0.02 mg/ml respectively. Moderate inhibition was noted with C. neoformans (MIC value of 0.5 mg/ml) and S. pyogenes (MIC value of 0.6 mg/ml). Strong inhibition was noted against S. pneumoniae where a MIC value of 0.2 mg/ml was obtained. The antimicrobial activity of E. globulus ethanol extracts against S. pyogenes is supported by a study by Caceres et al. (1991). The study found that E. globulus clearly inhibited the in vitro growth of S. aureus as well as S. pyogenes. Takahashi et al. (2004) tested the antimicrobial activity of E. globulus dichloromethane: methanol extracts on nine micro- organisms. The MIC value obtained against E. faecalis was 31 mg/l (0.031 mg/ml) which was consistent with that found in this study where a MIC value of 0.02 mg/ml was obtained. Khan et al. (2009a) recently studied the ethanol extracts of E. globulus against a number of pathogens including E. faecalis and two strains of K. pneumoniae. The extract was found to be inactive at the concentration tested against both strains of K. pneumoniae. However, against E. faecalis an MIC value of 3130 ?g/ml (3.13 mg/ml) was found. This activity noted was not as good as that reported in this study. This variation could be due to the type of extract tested was different or the strain of the organism was not the same. 122 Table 6.3 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra and E. globulus alone and in combination. Micro-organism MIC (mg/ml) ?FIC A. afra E. globulus A. afra with E. globulus A. afra with E. globulus (1:1) Interpretation K. pneumoniae NCTC 9633 6.3 3.0 2.0 0.5 Synergy M. catarrhalis ATCC 23246 4.7 0.01 1.1 5.8 Antagonism E. faecalis ATCC 29212 6.3 0.02 2.1 7.6 Antagonism C. neoformans ATCC 90112 1.2 0.5 0.8 1.1 Indifferent S. pneumoniae ATCC 49619 0.5 0.2 0.3 0.9 Additive S. pyogenes ATCC 8668 1.1 0.6 0.3 0.3 Synergy S. agalactiae ATCC 55618 3.0 1.0 1.0 0.7 Additive M. smegmatis (clinical) 1.7 1.5 0.5 0.3 Synergy *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. In the combination of A. afra with E. globulus dichloromethane: methanol extracts, MIC values were all below 3.0 mg/ml. Some antimicrobial activity was seen against K. pneumoniae and E. faecalis while moderate inhibition was noted against M. catarrhalis and S. agalactiae. Of particular interest are the MIC values against C. neoformans, S. pneumoniae, S. pyogenes and M. smegmatis with MIC?s of 0.8, 0.3, 0.3 and 0.5 mg/ml respectively. These values were interpreted as strong inhibitory activity for the combination of A. afra with E. globulus dichloromethane: methanol extract. ?FIC values were calculated and only one of the combinations showed indifferent interactions i.e. against C. neoformans. Additive interactions were noted for the pathogens S. pneumoniae and S. agalactiae. Synergistic interactions were found against K. pneumoniae (?FIC value = 0.5), S. pyogenes and M. smegmatis with ?FIC values of 0.3. For the first time 123 in the study, antagonism was noted and seen against M. catarrhalis and E. faecalis with very high ?FIC values (5.8 and 7.6). If one examines the very low MIC values of E. globulus independently against M. catarrhalis and E. faecalis and then examines the combined efficacy, it is clear that when treating infections from these two pathogens it is not advisable to combine with A. afra. Looking at the traditional method of administration of the combination when it is administered through inhalation of the steam from the infusions or decoctions are administered (Hutchings et al., 1996). The alcohol extracts are not cited as being used traditionally this shows that care should be taken when using the combinations as the type of infection is not known to a traditional healer. Proper diagnostic tests should be considered to isolate the pathogen responsible for the infection prior to treatment. This antagonism seen could cause unwanted side effects and toxicity, or the plant extracts could be potentially harmful. Further investigations in this regard are recommended. 6.2.2.1.3 Aqueous extracts The aqueous extracts of E. globulus, as shown in Table 6.4, gave MIC values between 0.8 - 14.0 mg/ml against all the pathogens tested. Very poor activity was noted against S. pneumoniae with a MIC value of 14.0 mg/ml. Some antimicrobial activity was seen against K. pneumoniae, M. catarrhalis, E. faecalis and M. smegmatis. Moderate inhibition was obtained against S. pyogenes and S. agalactiae and noteworthy antimicrobial activity was seen against C. neoformans with a MIC value of 0.8 mg/ml. In the combination with A. afra, antimicrobial activities ranged between 0.4-8.0 mg/ml against the pathogens tested. Moderate inhibition was seen, with an MIC value of 1.0 mg/ml, against S. agalactiae. Some activity was observed in combination against pathogens C. neoformans, S. pneumoniae and S. agalactiae. Noteworthy strong inhibition was obtained against C. neoformans with a MIC value of 0.4 mg/ml. ?FIC values determined revealed no antagonism in the combination against any of the pathogens tested. Synergy was obtained against C. neoformans and S. pneumoniae (?FIC values of 0.3 and 0.2). Additive interactions were noted against K. pneumoniae, M. catarrhalis, S. pyogenes, S. agalactiae and M. smegmatis. Against E. faecalis, an indifferent pharmacological interaction was noted. 124 Table 6.4 MIC and ?FIC values of the aqueous extracts of A. afra and E. globulus alone and in combination. Micro-organism MIC (mg/ml) ?FIC A. afra E. globulus A. afra with E. globulus A. afra with E. globulus (1:1) Interpretation K. pneumoniae NCTC 9633 12.0 6.0 8.0 1.0 Additive M. catarrhalis ATCC 23246 8.0 3.5 4.0 0.8 Additive E. faecalis ATCC 29212 7.0 2.5 4.0 1.1 Indifferent C. neoformans ATCC 90112 4.0 0.8 0.4 0.3 Synergy S. pneumoniae ATCC 49619 8.0 14.0 2.0 0.2 Synergy S. pyogenes ATCC 8668 5.3 1.3 2.0 0.9 Additive S. agalactiae ATCC 55618 1.7 1.5 1.0 0.6 Additive M. smegmatis (clinical) 8.0 4.0 4.0 0.8 Additive *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. 6.2.2.2 Isobologram interpretation The essential oils (Figure 6.4 and inset) of the combination of A. afra with E. globulus against M. catarrhalis showed additive interactions for all the ratios tested. The dichloromethane: methanol extracts were indifferent at four ratios tested and completely antagonistic for five of them (Figure 6.4). The ratios in which antagonism were noted are at 8:2, 7:3, 6:4, 5:5 and 4:6. A. afra and E. globulus vary in concentration at these ratios, but the general trend suggests that when the two extracts come closer in concentration to each other (1:1), antagonism is the result. In contrast, the aqueous extracts (Figure 6.4 and inset) were synergistic at all ratios tested except one. The one exception was the 1:1 ratio and it showed an additive interaction. This is interesting to note as the traditional use is the inhalation of the steam of an infusion or a decoction of the combination is taken (Hutchings et al., 1996). 125 Furthermore, results of aqueous extracts against M. catarrhalis demonstrate that 1:1 combinations may not always be the best selection. Figure 6.4 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against M. catarrhalis; ? 1:1 combination. For K. pneumoniae (Figure 6.5), the essential oils of the combination showed additive interactions for all the ratios tested. The dichloromethane: methanol extracts showed additive interactions for most of the ratios. However, two exceptions at ratios 4:6 and 5:1 (Figure 6.5, a) were synergistic. This synergy occurred when A. afra and E. globulus were at the same ratio and when E. globulus was slightly more than A. afra (ratio 4:6). The aqueous extracts of the combination of A. afra with E. globulus showed a mixture of indifferent and additive interactions. Additivity was noted at ratios 6:4, 4:6, 3:7, 2:8 and 1:9 and the 1:1 combination. Indifferent interactions were seen at ratios 9:1, 8:2 and 7:3. A higher concentration of E. globulus is better to obtain more synergy in the combination of the aqueous extracts against K. pneumoniae. 0 1 2 3 4 0 1 2 3 4 5 6 7 8 9 4.5 6.0 7.5 9.0 M. catarrhalis ATCC 23246 MIC Aa in combination/MIC Aa independently M IC E g in co m b in at io n /M IC E g independentl y 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 MIC Aa in combination/MIC Aa independently M IC E g in co m bi na ti on /M IC E g in de pe nd en tl y 126 0.0 0.5 1.0 1.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 3.5 4.0 4.5 5.0 E. faecalis ATCC 29212 MIC Aa in combination/MIC Aa independently MI C E g in co m bi na ti on /M IC E g independentl y .0 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 MIC Aa in combination/MIC Aa independently M IC E g in co m bi na ti on /M IC E g in d ep en d en tl y b Figure 6.5 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against K. pneumoniae (a - ratios 4:6 and 5:1); ? 1:1 combination. In Figure 6.6, the interactions of the combination of A. afra with E. globulus against E. faecalis is shown. The essential oil (Figure 6.6, inset) results show additive interactions for all the ratios tested. Figure 6.6 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against E. faecalis (b ? ratios 6:4 and 1:1); ? 1:1 combination. 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 K. pneumoniae NCTC 9633 MIC Aa in combination/MIC Aa independently M IC E g in co m b ina ti on /M IC E g in d ep en d en tl y a 127 The dichloromethane: methanol extracts (Figure 6.6) showed additive interactions for three ratios (3:7, 2:8 and 1:9), where E. globulus is the dominant essential oil in the combination. Four of the ratios were indifferent and two ratios were antagonistic Figure 6.6 shows the antagonistic ratios at (b), which are the 6:4 and the 5:5 combinations of the essential oils. The aqueous extracts showed indifferent interactions for all the ratios tested, except one, which was additive (ratio 4:6). The isobolographic interactions of the combination against C. neoformans (Figure 6.7) show additive interaction for the essential oils at all the ratios tested. The dichloromethane: methanol extracts showed mixed interactions. Ratios 9:1, 8:2, 4:6, 1:9 and 3:7 show additive interactions while ratios 7:3, 6:4, 2:8, and the 1:1 combinations were indifferent. The aqueous extracts displayed very interesting activity with all the ratios to be synergistic. Figure 6.7 Isobologram of the combination of A. afra and E. globulus essential oils ( ), dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against C. neoformans; ? 1:1 combination. The interactions of the 1:1 combination evaluated using the ?FIC method and the isobologram interaction method were in congruence with each other against M. catarrhalis and E. faecalis. Variation was noted against K. pneumoniae with the dichloromethane: methanol extracts where the ?FIC value indicated additivity and the isobologram was synergistic. The aqueous extracts against K. pneumoniae also expressed some variation where 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 C. neoformans ATCC 90112 MIC Aa in combination/MIC Aa independently M IC E g in co m bi na ti on /M IC E g in de pe nd en tl y 128 the ?FIC interpretation was indifferent and the isobologram interpretation was additive. The essential oil results of the interactions were congruent. Against C. neoformans, the variations occurred in the essential oil 1:1 combination wherein the ?FIC interpretation was synergistic and the isobologram interpretation was additive. The dichloromethane: methanol extract combination showed additivity when the ?FIC was calculated and indifference with the isobologram. The interactions observed for the 1:1 combination against M. catarrhalis, E. faecalis, K. pneumoniae and C. neoformans using the ?FIC determination method were in congruence with those observed when using the isobologram method. The combination of E. globulus essential oil with chlorhexidine dicgluconate has been studied by Karpanen et al. (2008). The MIC chequerboard method was used to evaluate the antimicrobial activity against two strains of S. epidermidis of the combination, in suspension and in biofilms. Using the ?FIC determination method, the interactions of the combination in suspension was indifferent. However, when E. globulus was combined with chlorhexidine dicgluconate against both strains of S. epidermidis growing in biofilms, synergistic activity was obtained. It is fascinating to note that the interactions of the combination, whether evaluated by MIC, ?FIC or the isobolograms, showed that the essential oil were mostly additive. In Low et al. (1974) (Hasegawa et al., 2008), the essential oils of Eucalyptus spp is used for the treatment of pulmonary infections by inhalation. This is also the case in the combination of A. afra with E. globulus. The traditional use is cited in Hutchings et al. (1996) wherein it states that the combination is administered by the inhalation of the steam of the infusion. The results obtained in the study show the essential oils exhibit the most consistent activity with predominantly additive interactions against the four pathogens tested. Thus, correlates with the traditional use of the combination by traditional healers. The dichloromethane: methanol extracts in combination however, were the most antagonistic in their interactions against M. catarrhalis. Additivity or indifference was predominant against E. faecalis, K. pneumoniae and C. neoformans. 129 The aqueous extracts showed very good activity and if not synergistic interactions the ratios were additive against M. catarrhalis, K. pneumoniae and C. neoformans. Indifference was noted from three ratios against K. pneumoniae and against E. faecalis. These results are interesting as none of the combinations exhibited antagonism against the pathogens tested. In addition, the literature cites the combination to be taken as decoction (Hutchings et al., 1996). Thus, the traditional method of administration ties in with the results obtained. This serves to validate the use of A. afra in combination with E. globulus essential oils and aqueous extract to treat respiratory infections. 6.3 Conclusions ? The major compounds present in E. globulus were 1,8-cineole (63%) and ?-pinene (16.7%), limonene (3.6%) and ?-cymene (2.7%). ? The essential oil of E. globulus showed some antimicrobial activity with the best and noteworthy activity against C. neoformans (MIC value of 0.6 mg/ml). ? The dichloromethane: methanol extract of E. globulus showed very good antimicrobial activity against, C. neoformans (MIC value = 0.5 mg/ml), S. pneumoniae (MIC value = 0.19 mg/ml) and S. pyogenes (MIC value = 0.56 mg/ml) with the best activity against E. faecalis with a MIC value of 0.02 mg/ml and M. catarrhalis (MIC value = 0.014 mg/ml). ? In the combination of the dichloromethane: methanol extracts of A. afra with E. globulus, the best antimicrobial activity was obtained against S. pneumoniae (MIC value = 0.25 mg/ml), S. pyogenes (MIC value = 0.25 mg/ml) and M. smegmatis (MIC value = 0.5 mg/ml). ? The ?FIC determination method of the dichloromethane: methanol extract combination gave antagonistic interactions against M. catarrhalis and E. faecalis. Synergy was noted against K. pneumoniae, S. pyogenes and M. smegmatis. ? The best MIC value obtained with the aqueous extract of E. globulus was 0.8 mg/ml against C. neoformans. The combination with A. afra also gave the best MIC value against C. neoformans (MIC value of 0.4 mg/ml). ? The combination of the aqueous extracts showed synergistic ?FIC interactions against C. neoformans (0.3) and S. pneumoniae (0.2) 130 ? The essential oils of the combination of A. afra with E. globulus showed additive interactions against M. catarrhalis, K. pneumoniae, E. faecalis and C. neoformans when assessing the isobolograms. ? The isobolograms showed antagonistic interactions (five ratios) for the dichloromethane: methanol extract of the combination against M. catarrhalis. The aqueous extracts of the combination showed very promising results with most or all ratios showing synergy against M. catarrhalis and C. neoformans. ? Overall, C. neoformans was the most sensitive pathogen to the combination of A. afra with E. globulus essential oils and aqueous extracts. 131 Chapter 7 The antimicrobial efficacy of Artemisia afra Jacq. ex Willd and Zanthoxylum capense (Thunb.) Harv. in combination 7.1 Introduction The combination of A. afra with Z. capense has been used by traditional healers for the treatment of head colds and influenza (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). It was a popular remedy in the influenza epidemic in 1918. A decoction and an infusion of the leaves of Z. capense is made with A. afra and cited as been used in febrile conditions. If it was used as a remedy for influenza since 1918, it follows that the combination be analyzed for its validity in treatment of respiratory illnesses. Even though Z. capense is aromatic in nature, no essential oil was obtained through hydro-distillation. The dichloromethane: methanol extracts and the aqueous extracts were thus only studied. 7.2 Results and discussion 7.2.1 Chromatographic techniques 7.2.1.1 Thin layer chromatography Thin layer chromatography analysis of the dichloromethane: methanol extracts of Z. capense revealed at 254 nm (Figure 7.1), that many of the compounds did not migrate up the plate. It is also evident at 365 nm that a blue fluorescent band was visible at the bottom. Many fluorescent red compounds are also visible at 365 nm. In Z. capense alone and in the combination with A. afra, a dark green band is visible at Rf = 0.9. A bright yellow compound at Rf = 0.8 is also seen under white light (Figure 7.1). After derivatization, the compounds mentioned are seen as bluish-purple. Three fluorescent compounds seen at 365 nm are dominant in the combination of A. afra with Z. capense dichloromethane: methanol extracts. These are shown in Figure 7.1. two compounds are from A. afra and extract 1 compound is from Z. capense extract. The aqueous extracts as seen in Figure 7.2, showed a limited number of compounds at 254 nm. 132 Figure 7.1 A. afra dichloromethane: methanol extracts alone and in combination with Z. capense visualizing at 254 nm, 365 nm, white light and after derivatization with vanillin- sulphuric acid. Aa - A. afra; Zc - Z.capense; Aa + Zc - A. afra with Z. capense. Figure 7.2 A. afra aqueous extracts alone and in combination with Z. capense visualizing at 254 nm, 365 nm, white light and after derivatization with vanillin-sulphuric acid. Aa - A. afra; Zc - Z.capense; Aa + Zc - A. afra with Z. capense. 7.2.2 Antimicrobial analysis 7.2.2.1 MIC assays and FIC determination 7.2.2.1.1 Dichloromethane: methanol extracts For the dichloromethane: methanol extracts (Table 7.1), moderate inhibitory antimicrobial activity was noted against E. faecalis and S. pneumoniae. Some antimicrobial activity was noted against M. catarrhalis, K. pneumoniae, S. pyogenes and S. agalactiae. Noteworthy 254 nm 365 nm White light Derivatized Aa Zc Aa + Zc Aa Zc Aa + Zc Aa Zc Aa + Zc Aa Zc Aa + Zc 254 nm Derivatized 365 nm White light Aa Zc Aa + Zc Aa Zc Aa + Zc Aa Zc Aa + Zc Aa Zc Aa + Zc 133 activity with strong inhibition was obtained against C. neoformans (MIC value = 0.4 mg/ml) and moderate inhibition against M. smegmatis with MIC values of 0.8 mg/ml. Table 7.1 MIC and ?FIC values of A. afra and Z. capense dichloromethane: methanol extracts independently and in combination. *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Antimicrobial activity (moderate inhibition) was noted for the combination of A. afra with Z. capense dichloromethane: methanol extracts, against S. pyogenes with an MIC value of 1.0 mg/ml. Some antimicrobial activity was seen against K. pneumoniae, M. catarrhalis, E. faecalis, S. agalactiae and M. smegmatis. The lowest MIC values and noteworthy inhibition were obtained against pathogens C. neoformans (0.4 mg/ml) and S. pneumoniae (0.3 mg/ml) ?FIC values of the combination of the dichloromethane: methanol extracts of A. afra with Z. capense demonstrated synergy against S. pneumoniae (?FIC value = 0.4). Additive ?FIC values were obtained for the combination against K. pneumoniae, M. catarrhalis, E. faecalis, Micro-organism MIC (mg/ml) ?FIC A. afra Z. capense A. afra with Z. capense A. afra with Z. capense (1:1) Interpretation K. pneumoniae NCTC 9633 6.3 1.7 2.0 0.8 Additive M. catarrhalis ATCC 23246 4.7 2.0 2.0 0.7 Additive E. faecalis ATCC 29212 6.3 1.3 2.0 0.9 Additive C. neoformans ATCC 90112 1.2 0.4 0.4 0.7 Additive S. pneumoniae ATCC 49619 0.5 1.5 0.3 0.4 Synergy S. pyogenes ATCC 8668 1.1 2.0 1.0 0.7 Additive S. agalactiae ATCC 55618 3.0 4.0 4.0 1.2 Indifference M. smegmatis (clinical) 1.7 0.8 2.0 1.9 Indifference 134 C. neoformans and S. pyogenes. Indifferent interactions were noted for the combination against S. agalactiae and M. smegmatis. 7.2.2.1.2 Aqueous extracts The aqueous extracts of Z. capense (Table 7.2), showed the lowest MIC value with strong and noteworthy activity against S. agalactiae at 0.4 mg/ml. Some antimicrobial activity was seen against C. neoformans with a MIC value of 2.0 mg/ml. Table 7.2 MIC and ?FIC of A. afra and Z. capense aqueous extracts independently and in combination. *Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for Z. capense/A. afra with Z. capense was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml) ?FIC A. afra Z. capense A. afra with Z. capense A. afra with Z. capense (1:1) Interpretation K. pneumoniae NCTC 9633 12.0 13.3 12.0 1.0 Additive M. catarrhalis ATCC 23246 8.0 16.0 8.0 0.8 Additive E. faecalis ATCC 29212 7.0 16.0 ?16.0 ND1 Additive/Antagonistic2 C. neoformans ATCC 90112 4.0 2.0 4.0 1.5 Indifferent S. pneumoniae ATCC 49619 8.0 ?16.0 ?16.0 ND1 Additive/Antagonistic2 S. pyogenes ATCC 8668 5.3 ?16.0 ?16.0 ND1 Additive/Antagonistic2 S. agalactiae ATCC 55618 1.7 0.4 1.3 2.0 Indifferent M. smegmatis (clinical) 8.0 ?16.0 3.0 ND1 Synergy2 135 In 2003, Motsei et al. tested the aqueous and the solvent extracts (ethanol, ethyl acetate, and hexane extracts) of Z. capense against C. albicans clinical isolates from a 5-month-old baby, an adult, and a standard strain. Results showed that the aqueous extract and the solvent extracts of the bark of Z. capense to be inactive against all the isolates at the highest concentration (100 mg/ml) tested. In a study by Buwa and van Staden, (2006) that investigated the antimicrobial activity of South African traditional medicinal plants to treat venereal diseases, Z. capense leaf was one of the plants tested against B. subtilis, E. coli, K. pneumoniae, S. aureus as well as Candida albicans. The ethanol extract of Z. capense against K. pneumoniae yielded an MIC value of 3.125 mg/ml and the aqueous extract gave an MIC value of 12.5 mg/ml against the same pathogen (Buwa and van Staden, 2006). In the current study, results showed that against K. pneumoniae, Z. capense aqueous extract gave an MIC value of 13.3 mg/ml. This is consistent and comparable with the study conducted by Buwa and van Staden (2006). The combination of Z. capense with A. afra against S. agalactiae yielded the highest antimicrobial activity (1.3 mg/ml). Some activity was noted against C. neoformans and M. smegmatis and very poor activity was obtained against K. pneumoniae and M. catarrhalis. No inhibition was noted against E. faecalis, S. pneumoniae and S. pyogenes. ?FIC values calculated denoted indifferent interactions against S. agalactiae and C. neoformans and additivity against K. pneumoniae and M. catarrhalis. No end point MIC value was obtained for Z. capense against S. pneumoniae, S. pyogenes and M. smegmatis and in the combination against E. faecalis, S. pneumoniae and S. pyogenes. Thus, ?FIC values could not be calculated. However, tentative interpretations based on the MIC values obtained were considered. (Table 7.2) 7.2.2.2 Isobologram interactions For the combination of A. afra and Z. capense against M. catarrhalis (Figure 7.3), the dichloromethane: methanol extracts showed mainly additive interactions with the exception of two points at the ratio 2:8 and 1:9 (Figure 7.3, a), which proved indifferent. These points are ratios in which Z. capense is in the majority. For the aqueous extracts no end point MIC value was determined at the highest concentration tested thus no isobologram could be constructed. However, Figure 7.7 shows a graph depicting the MIC values of the combination 136 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 M. catarrhalis ATCC 23246 MIC Aa in combination/MIC Aa independently M IC Z c in co m bi na ti on /M IC Z c in de pe nd en tl y a at different ratios. The results show that the MIC value increases as the ratio of Z. capense aqueous extract increases. The MIC value remains the same as A. afra alone for ratios, 9:1, 8:2, and 7:3, and then it increases from the ratio 6:4 where no MIC value was obtained at the highest concentration tested. Thus, no increase in activity is seen when the aqueous extracts of A. afra was combined with Z. capense at different ratios. Figure 7.3 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) against M. catarrhalis (a ? ratio 2:8 and 1:9); ? 1:1 combination. The combination against K. pneumoniae as shown in Figure 7.4 yielded additive interactions for all the ratios tested for the dichloromethane: methanol extracts. Interactions of the aqueous extracts are given in Figure 7.7 where the MIC values of the different ratios are given against K. pneumoniae. The MIC values of the ratios 9:1, 8:2, 7:3, 6:4 and 5:5 are lower than the MIC values of A. afra and Z. capense individually. Thus, a tentative interpretation of synergy is made for the interaction of these ratios of the combination. The MIC values show additive interactions at ratios 4:6, 3:7 and 2:8. It is evident that an increasing ratio of Z capense leads to an increase in MIC value against K. pneumoniae. 137 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1.25 K. pneumoniae NCTC 9633 MIC Aa in combination/MIC Aa independently M IC Z c in co m bi na ti on /M IC Z c in de pe nd en tl y 0.00 0.25 0.50 0.75 1.00 1.25 0.00 0.25 0.50 0.75 1.00 1. E. faecalis ATCC 29212 MIC Aa in combination/MIC Aa independently M IC Z c in co m bi na ti on /M IC Z c in de pe nd en tl y b Figure 7.4 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) against K. pneumoniae; ? 1:1 combination. Isobologram interactions of the combination of A. afra and Z. capense against E. faecalis (Figure 7.5) showed mainly additive interactions for the dichloromethane: methanol extracts. One ratio was synergistic (Figure 7.5, b), i.e. 6:4, where A. afra was in the majority in the combination. Figure 7.5 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) against E. faecalis (b ? ratio 6:4); ? 1:1 combination. 138 The graph depicted in Figure 7.7 shows the MIC values of the combination at different ratios, against E. faecalis, increasing as the ratio of Z. capense increases. Additivity of the combination is seen at ratios 9:1, 8:2 and 7:3, when A. afra is the dominant extract present. From the 1:1 ratio, the MIC value is not determinable at the highest concentration tested. The dichloromethane: methanol extracts of the combination of A. afra with Z. capense against C. neoformans (Figure 7.6) showed additivity for most of the ratios tested. Synergistic activity was obtained for one ratio i.e. 7:3 (Figure 7.6, c), where A. afra was dominant. The aqueous extracts showed interactions including additivity and indifference. Two points, at ratios 4:6 and 1:9, where Z. capense was dominant, was additive. The remaining ratios tested gave indifferent pharmacological profiles. Figure 7.6 Isobologram of the combination of A. afra and Z. capense dichloromethane: methanol extracts ( ) and aqueous extracts ( ) against C. neoformans (c ? ratio 7:3); ? 1:1 combination. Against S. pneumoniae, S. pyogenes and M. smegmatis the aqueous extract of Z. capense was not active (Table 7.2). For the aqueous extracts of the combination of A. afra with Z. capense, MIC values (Table 7.2) were not determined at the highest concentration tested against E. faecalis, S. pneumoniae and S. pyogenes. Tentative interpretations were carried out to determine the ?FIC interpretations of the interactions. For the isobologram construction, the MIC values of Z. capense could not be calculated against M. catarrhalis, K. pneumoniae and 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 C. neoformans ATCC 90112 MIC Aa in combination/MIC Aa independently M IC Z c in co m bi na ti on /M IC Z c in de pe nd en tl y c 139 E. faecalis. Comparisons of the interpretations are made against the pathogens M. catarrhalis, K. pneumoniae and E. faecalis. Against M. catarrhalis, the ?FIC calculated (Table 7.2) was interpreted as additive and the tentative interpretation of the combination in ratios (Figure 7.7) was interpreted as being additive or indifferent. Against K. pneumoniae, ?FIC interpretation was additive and the tentative interpretation of the ratios (Figure 7.7) was synergistic or additive. The ?FIC value and the interpretation of the ratios (Figure 7.7) against E. faecalis could not be determined, thus tentative interpretations were carried out and both were consistent and interpreted as either additive or antagonistic. Figure 7.7 The MIC values of the different ratios of the combination of A. afra with Z. capense aqueous extracts against K. pneumoniae, M. catarrhalis and E. faecalis. No other comparable studies are available to compare interactions noted between A. afra with Z. capense. The aqueous extracts of A. afra and Z. capense would represent an infusion or decoction. Z. capense proved inactive at the concentrations tested. However, other ways to determine if the combination is synergistic for the above-mentioned diseases or illness are available e.g. time-kill assays. Nevertheless, the isobologram results obtained give an indication that the dichloromethane: methanol extracts of Z. capense in combination with A. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 10:0 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9 0:10 M IC val u e (m g/ m l) Ratio (A. afra: Z. capense) K. pneumoniae M. catarrhalis E. faecalis >16 140 afra, give synergistic and additive results depending on the ratios in which they are combined. 7.3 Conclusions ? TLC of Z. capense dichloromethane: methanol extracts revealed many different compounds present. Thus in the combination of A. afra and Z. capense the number of compounds present increased. ? The best antimicrobial activities noted for Z. capense dichloromethane: methanol extracts individually was against C. neoformans (MIC value = 0.4 mg/ml) and M. smegmatis (MIC value = 0.8 mg/ml). ? Noteworthy synergistic activity was noted in the combination of A. afra with Z. capense dichloromethane: methanol extract against S. pneumoniae (MIC value of 0.3 mg/ml and ?FIC value of 0.4). ? The highest antimicrobial activity (MIC value of 0.4 mg/ml) obtained for the aqueous extracts of Z. capense was against S. agalactiae. ? The combination of A. afra with Z. capense aqueous extracts also yielded the best MIC value against S. agalactiae (MIC value = 1.3 mg/ml). ? Isobologram interactions revealed mainly additivity for the dichloromethane: methanol extracts against the four pathogens tested. Synergy was noted in the ratio 6:4 against K. pneumoniae and 7:3 against C. neoformans. 141 Chapter 8 Artemisia afra in triple plant combinations 8.1 Introduction Western (conventional) medicine has increasingly begun using three or more agents as viable antimicrobial treatment options (Lange, 1995). This has been achieved by combining drugs like anti-retrovirals (Stavudine?-Lamivudine?-Efavirenz?), analgesics (Synap Forte?, Lentogesic?), as well as chemotherapeutic drugs (Irinotecan-fluorouracil-folinic acid) (Rossiter et al., 2010; Turner et al., 2010). A study by Ocio et al. (2010), has confirmed the efficacy of a triple combination in contrast to a double combination for the treatment of multiple myeloma. Examples of studies incorporating triple combinations include those discussed by Rochon-Edouard et al. (2000) which investigated the efficacy of ?-lactams, vancomycin and netilmicin against methicillan-resistant S. aureus strains using time-kill studies. Results showed that the combination could, at sub-inhibitory concentrations be bactericidal after 24 hrs. These are at higher rates than a double combination of vancomycin- netilmicin. Another study of note is by O?Shaughnessy et al. (2006), where the antifungal interaction of amphotericin B, caspofungin and voriconazole was tested in combination at different drug concentrations, against Aspergillus species. Antifungal interactions, namely synergy, was found at low concentrations of amphotericin B (<0.2 mg/l) and voriconazole (<0.5 mg/l). Antagonism was found when the concentrations of amphotericin B and voriconazole was increased i.e. between 0.3-0.5 mg/l and >0.25 mg/l respectively. Another study pertaining to triple combinations and their uses for the treatment of Plasmodium falciparum infections, demonstrated no decrease in activity when folinic acid was added to a double combination (Yeo and Rieckmann, 1997). The third compound could possibly alleviate toxic effects of the other two compounds and thus prevent adverse effects normally seen in the double combination. In order to fully explore the use of combinations in traditional medicine, it is important to recognise that plants are not only used in a two-plant combination. There are instances where three or more plants are used. Double combinations have been discussed in detail in Chapters 3-7. Typically, one would assume that the addition of a third substance to a combination would increase or improve the efficacy of a combination. However, the combinations of substances in triple or more cannot be assumed to result in synergistic or even additive 142 interaction. The combinations must be verified and the interaction must be established (O?Shaughnessy et al., 2006). Natural drugs or compounds are known to exhibit complex pharmacological action (Biavatti, 2009), therefore, the outcome of combining more than one or more plants is difficult to predict. Thus, it is important to test the activity in combination and thereafter have a basis upon which we may be able to scientifically validate the combinations in which they are prescribed. Triple combinations of ethnobotanical origin are used substantially all over the world for the treatment of various conditions. Figure 8.1 shows the results obtained from an internet search on the use of triple plant combinations in general and then on triple combinations of plants in South Africa. The findings show that there is little scientific evidence on the validation of the use of triple combinations, particularly for Southern African plants. Only one validation based on southern African plants could be found, Sibandze et al., (2010), combined three Swazi traditional medicinal plants i.e. Breonadia salicina, Syzgium cordatum with Ozoroa sphaerocarpa to treat diarrhoea. Results of this study showed predominantly synergism when the three plants were combined and thus support the traditional use of the combination in Swazi traditional medicine. Other triple plant combination studies elsewhere in the world include the investigation of corni fructus, cinnamon and Chinese chive to determine the antimicrobial efficacy of the three plant extracts in combination against common food borne pathogens (Hsieh et al., 2001). Results showed that the combination 8:1:1 of the extracts produced an entire spectrum of antimicrobial activity as well as being very stable to heat and pH changes. In South African traditional medicine, combinations of plants, either in triple or more are used frequently by traditional healers (Watt and Breyer-Brandewijk, 1962; Hutchings et al., 1996; Felhaber, 1997). Some such examples are seen in Table 1.3 and include the boiling and drinking of the combination of the powdered leaves of Eucalyptus globulus with powdered root of Corbichonia decumbens and powdered rhizome of A. calamus to relieve a stuffy or blocked nose. Phoenix reclinata, Euclea natalensis, Capparis tomentosa and Maytennus heterophylla are combined to treat pleurodynia and pleurisy wherein the steam from the decoction is blown into the wound (Bryant, 1970; Hutchings et al., 1996). 143 Figure 8.1 Summary of the reports available on triple combinations of medicinal plants. General search on conventional antimicrobial and ethnobotanical combinations "Triple combinations" Search for triple combinations within ethnobotanicals "Triple combinations AND plants" About 5,580,000 results using ?Google? 13,813 results using ?ScienceDirect? 417 results on ?Scopus? 50 hits on ?PubMed? Ethnobotanically in SA "Triple combinations AND plants AND South Africa" 1356 hits on ?ScienceDirect? 15 results on ?Scopus? 1 result on ?Pubmed? About 1,100,000 results using ?Google? 95,280 results using ?ScienceDirect? 8722 results using ?Scopus? 1517 using ?PubMed? About 2,520,000 results using ?Google? 144 A boiled infusion of the combination of Eucalyptus globulus, Corbichonia decumbens and A. calamus is also used for a blocked nose (Felhaber, 1997). Combinations where A. afra is a component are in the treatment of asthma, wherein the root of A. amatymbica with the leaves of A. afra and the leaves of L. leonurus are boiled together, and taken (Felhaber, 1997). For the treatment of chronic bronchitis and emphysema, the leaves of A. afra are mixed with the bark of W. salutaris and the rhizome of Acoras calamus, which are then boiled and drank (Felhaber, 1997). The leaves of A. afra, O. asteriscoides with E. globulus were reported to be used extensively by the Europeans in South Africa for the treatment of influenza. An infusion of the leaves of A. afra, Leonotis microphylla and E. globulus is used by the Sotho and European people in South Africa for the relief of digestive disturbances accompanied by fever as well as for chest infections. The decoction of the combination of A. afra, Allium sativum with Zanthoxylum capense is used as a febrifuge (Watt and Breyer-Brandewijk, 1962). From the literature searched, none of the afore-mentioned combinations have been scientifically validated. Moreover, evidence for synergy or antagonism between three or more plant combinations is sparse. It is important that these combinations are properly validated in order to reduce complications and adverse effects that might result if the plants used together are incompatible. Four of the triple combinations mentioned in Table 1.3 that included A. afra in the treatment of respiratory diseases have been selected for this study. The selection was based on the availability of the plant species, keeping in mind sustainable harvesting practices. The triple combinations evaluated are A. afra, O. asteriscoides with E. globulus; A. afra, L. randii with E. globulus and the combination of A. afra, Allium sativum with Z. capense. 8.2 Results and discussion 8.2.1 A. afra, O. asteriscoides and E. globulus in combination The botanical description, locality, medicinal uses and phytochemistry of A. afra is discussed in Chapter 1 and similarly for E. globulus and O. asteriscoides are discussed in Appendix A4 and A7, respectively. The dichloromethane: methanol extracts of A. afra, O. asteriscoides and E. globulus were evaluated using thin layer chromatography. MIC determination and FIC 145 interpretation was carried out on the essential oils, dichloromethane: methanol and the aqueous extracts according to the methods described in Chapter 2. 8.2.1.1 Chromatographic techniques 8.2.1.1.1 Thin layer chromatography The compounds present in the plant extracts at 256 nm, individually and in combination fluoresce somewhat blackish under this wavelength due to the presence of conjugated double bonds (Figure 8.2). 1 A. afra 2 O. asteriscoides 3 E. globulus 4 A. afra with O. asteriscoides 5 A. afra with E. globulus 6 O. asteriscoides with E. globulus 7 A. afra, O. asteriscoides with E. globulus 1 2 3 4 5 6 7 1 2 3 4 5 6 7 254 nm White light 1 2 3 4 5 6 7 1 2 3 4 5 6 7 365 nm 146 Figure 8.2 TLC plates of the dichloromethane: methanol extracts of A. afra, O. asteriscoides and E. globulus alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. At 365 nm (Figure 8.2), some fluorescent compounds, bright blue and red compounds, are visible. For the TLC plate viewed under white light, only the coloured compounds are detected i.e. a green compound at Rf = 0.9 in A. afra. When the TLC plate was derivatized with vanillin-sulphuric acid reagent, organic compounds in the test sample are coloured yellow, brown, or black (Houghton and Raman, 1998). The combination of the dichloromethane: methanol extracts of A. afra, O. asteriscoides and E. globulus independently and in combination thus yield compounds that are visible under UV light at 254 nm and 365 nm. 8.2.1.2 Antimicrobial analysis 8.2.1.2.1 MIC assays and FIC determination MIC values of A. afra, O. asteriscoides and E. globulus individually and in combination are included in Table 8.1, 8.2 and 8.3 for the sake of completeness, (shaded areas) but have been discussed in Chapters 3, 4 and 6. The combination of O. asteriscoides with E. globulus has been studied here to give a detailed overview of each 1:1 plant combination within the triple combination. MIC values for the controls i.e. ciprofloxacin and amphotericin B are shown in Table 3.3. 8.2.1.2.1.1 Essential oils Table 8.1 shows the MIC values of O. asteriscoides in combination with E. globulus essential oils against eight pathogens. The highest antimicrobial activity with noteworthy inhibition Derivatized 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 147 was obtained for the combination of O. asteriscoides with E. globulus against E. faecalis, (0.5 mg/ml). Other noteworthy activity was noted for the combination against C. neoformans (MIC value = 0.75 mg/ml), S. pneumoniae (MIC value = 1.0 mg/ml), S. pyogenes (MIC value = 2.0 mg/ml) and M. smegmatis (MIC value = 1.5 mg/ml). Some antimicrobial activity was obtained in the combination against S. agalactiae and very low activity against K. pneumoniae and M. catarrhalis. ?FIC values calculated for the combination of O. asteriscoides with E. globulus showed additive interactions against K. pneumoniae, M. catarrhalis, C. neoformans S. pneumoniae, S. agalactiae and M. smegmatis. The most significant synergistic activity in this triple combination study was noted against E. faecalis with a ?FIC value = 0.1. In the combination of the three essential oils i.e. A. afra, O. asteriscoides and E. globulus, noteworthy activity was obtained against S. pneumoniae, S. pyogenes and M. smegmatis (MIC value = 2.0 mg/ml). Some activity was noted against S. agalactiae and very poor activity was seen against K. pneumoniae, M. catarrhalis, E. faecalis and C. neoformans. ?FIC?s that were then calculated and demonstrated antagonistic activity against C. neoformans (?FIC value = 6.5). Additive interactions were noted against K. pneumoniae, M. catarrhalis, E. faecalis and S. agalactiae. Against S. pneumoniae, S. pyogenes and M. smegmatis ?FIC values showed indifferent/non-interactive interactions (?FIC values 1.1- 1.3) (Table 8.1). In the combination of A. afra with O. asteriscoides essential oils, six of the pathogens showed additive interactions and two pathogens showed indifference. The combination of A. afra with E. globulus, all eight pathogens tested showed additive pharmacological interactions. When O. asteriscoides was combined with E. globulus, six pathogens were additive, one was indifferent and the interaction against E. faecalis was synergistic. When A. afra, O. asteriscoides and E. globulus essential oils were combined in a 1:1:1 ratio, four pathogens showed additive interactions, three were indifferent and one pathogen showed antagonism (C. neoformans). 148 Table 8.1 MIC and ?FIC values of the essential oils of A. afra, O. asteriscoides and E. globulus alone and in combination. *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3, 4 and 5 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml)/ ?FIC value indicated in brackets where applicable ?FIC A. afra O. asteriscoides A. afra with O. asteriscoides E. globulus A. afra with E. globulus O. asteriscoides with E. globulus A. afra, O. asteriscoide s with E. globulus A. afra, O. asteriscoides with E. globulus Interpretation K. pneumoniae NCTC 9633 8.0 8.0 8.0 (1.0) 8.0 8.0 (1.0) 8.0 (1.0) 8.0 1.0 Additive M. catarrhalis ATCC 23246 8.7 8.0 8.0 (1.0) 8.0 8.0 (1.0) 8.0 (1.0) 8.0 1.0 Additive E. faecalis ATCC 29212 8.7 8.0 8.0 (1.0) 8.0 8.0 (1.0) 0.5 (0.1) 8.0 1.0 Additive C. neoformans ATCC 90112 3.8 2.0 2.0 (0.8) 0.6 0.8 (0.8) 0.8 ( 0.8) 8.0 6.5 Antagonism S. pneumoniae ATCC 49619 3.3 1.0 1.0 (0.7) 2.7 3.0 (1.0) 1.0 (0.7) 2.0 1.1 Indifferent S. pyogenes ATCC 8668 1.7 1.7 1.5 (0.9) 2.0 1.5 (0.8) 2.0 (1.1) 2.0 1.1 Indifferent S. agalactiae ATCC 55618 4.8 4.7 7.0 (1.5) 5.3 4.0 (0.8) 4.0 (0.8) 4.0 0.8 Additive M. smegmatis (clinical) 1.5 1.3 1.5 (1.1) 2.0 1.3 (0.7) 1.5 (1.0) 2.0 1.3 Indifferent 149 The triple combination did not show any marked increase in activity or synergy when compared to the double combinations. In fact, against C. neoformans, it is imperative that the three oils are not combined as detrimental effects could result due to the antagonism they present. Inhalation is not used when administering this combination thus it was not surprising that the combination did not show any marked improvement in interaction. One combination that stands out in the combinations involving the essential oils is O. asteriscoides with E. globulus against E. faecalis where the most significant synergistic interaction was observed for the entire study. 8.2.1.2.1.2 Dichloromethane: methanol extracts For the dichloromethane: methanol extracts, MIC values of the combination of O. asteriscoides with E. globulus are shown in Table 8.2. Noteworthy inhibitory activity was seen against C. neoformans (MIC value = 0.3 mg/ml), S. pneumoniae (MIC value = 0.5 mg/ml) and S. agalactiae (MIC value = 0.5 mg/ml). Moderate inhibition was noted against S. pyogenes with a MIC value of 1.0 mg/ml. Some antimicrobial activity was seen against K. pneumoniae, M. catarrhalis, E. faecalis and M. smegmatis. The ?FIC values calculated for the combination of O. asteriscoides with E. globulus gave additive interactions against K. pneumoniae and C. neoformans with ?FIC values of 0.7 and 0.8 respectively. Synergy was obtained against S. agalactiae where the ?FIC calculated was 0.4. Antagonism was observed for studies against M. catarrhalis and E. faecalis. For the triple combination of the plants extracts, MIC values (0.5 mg/ml) demonstrated noteworthy strong inhibition against C. neoformans and S. agalactiae. Noteworthy moderate inhibition was observed for S. pneumoniae (MIC value = 0.6 mg/ml). Moderate inhibitory antimicrobial activity was obtained against S. pyogenes with a MIC value of 1.0 mg/ml. Some activity was noted against K. pneumoniae, M. catarrhalis, E. faecalis and M. smegmatis. ?FIC values of the triple combination yielded synergistic interactions against two pathogens i.e. against K. pneumoniae (?FIC value = 0.5) and S. agalactiae (?FIC value = 0.4). Two pathogens revealed complete antagonism when the ?FIC values were calculated. These were M. catarrhalis and E. faecalis. One combination was additive against C. neoformans (?FIC value = 0.9). 150 Table 8.2 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, O. asteriscoides and E. globulus alone and in combination. *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3, 4 and 5 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml)/?FIC value indicated in brackets where applicable ?FIC A. afra O. asteriscoid es A. afra with O. asteriscoides E. globulus A. afra with E. globulus O. asteriscoid es with E. globulus A. afra, O. asteriscoides with E. globulus A. afra, O. asteriscoides with E. globulus Interpretation K. pneumoniae NCTC 9633 6.3 4.0 6.0 (1.2) 3.0 2.0 (0.5) 2.0 (0.7) 2.0 0.5 Synergism M. catarrhalis ATCC 23246 4.7 4.0 4.0 (0.9) 0.01 1.1 (5.8) 2.2 (77.3) 4.0 57.6 Antagonism E. faecalis ATCC 29212 6.3 2.0 4.0 ( 1.3) 0.02 2.1 (7.6) 1.1 ( 34.0) 2.0 42.9 Antagonism C. neoformans ATCC 90112 1.2 0.3 0.3 ( 0.7) 0.5 0.8 ( 1.1) 0.3 ( 0.8) 0.5 0.9 Additive S. pneumoniae ATCC 49619 0.5 0.4 0.1 (0.3) 0.19 0.3 (0.9) 0.5 (1.9) 0.6 1.9 Indifferent S. pyogenes ATCC 8668 1.1 0.4 0.1 (0.2) 0.6 0.3 (0.3) 1.0 (2.1) 1.0 1.7 Indifferent S. agalactiae ATCC 55618 3.0 1.3 1.0 (0.6) 1.0 1.0 (0.7) 0.5 (0.4) 0.5 0.4 Synergism M. smegmatis (clinical) 1.7 1.4 0.5 (0.3) 1.5 0.5 (0.3) 2.0 (1.4) 2.0 1.3 Indifference 151 The combination of the dichloromethane: methanol extracts of A. afra with O. asteriscoides showed varied interactions .The combination of A. afra with E. globulus demonstrated three synergistic interactions (against K. pneumoniae, S. pyogenes and M. smegmatis) and two antagonistic (M. catarrhalis and E. faecalis) interactions.. For the combination of O. asteriscoides with E. globulus, only one interaction was evident and this was against S. agalactiae. The triple combination of A. afra with O. asteriscoides and E. globulus demonstrated varied interactions with two synergistic interactions against K. pneumoniae and S. agalactiae. The triple combination is thus better than the double combinations when treating K. pneumoniae and S. agalactiae relating infections. However, care should be taken when treating M. catarrhalis and E. faecalis infections due to the antagonism encountered with these two pathogens. Thus, the causative micro-organism should be identified before treatment is administered to avoid any unwanted side effects. 8.2.1.2.1.3 Aqueous extracts The MIC values of the aqueous extracts (Table 8.3) of the combination of O. asteriscoides with E. globulus demonstrated moderate inhibition against S. agalactiae (MIC value = 1.0 mg/ml). Some antimicrobial activity was obtained against M. catarrhalis, E. faecalis, C. neoformans, S. pneumoniae, S. pyogenes and M. smegmatis. The aqueous extracts showed additive ?FIC values of 0.8, 1.0, 0.7 and 1.0 against M. catarrhalis, E. faecalis, S. agalactiae and M. smegmatis respectively. Against K. pneumoniae, C. neoformans, S. pneumoniae and S. pyogenes indifferent interactions were noted. The MIC values of the triple combination ranged from 2.0 - 6.0 mg/ml with K. pneumoniae having the least sensitivity to the combination (MIC value of 6.0 mg/ml). In contrast, the triple combination was most active against S. pyogenes and S. agalactiae with MIC values of 2.0 mg/ml. The ?FIC values of the triple combination against K. pneumoniae, M. catarrhalis, E. faecalis, S. pyogenes and M. smegmatis all demonstrate additive interactions i.e. ?FIC values of 0.8, 0.7, 0.9, 1.0 and 0.8 respectively. ?FIC values calculated against C. neoformans, S. pneumoniae and S. agalactiae showed indifferent interactions when the aqueous extracts of all three plants were combined. 152 Table 8.3 MIC and ?FIC values of the aqueous extracts of A. afra, O. asteriscoides and E. globulus alone and in combination. Micro-organism MIC (mg/ml)/?FIC value indicated in brackets where applicable ?FIC A. afra O. asteriscoides A. afra with O. asteriscoide s E. globulus A. afra with E. globulus O. asteriscoides with E. globulus A. afra, O. asteriscoides with E. globulus A. afra, O. asteriscoides with E. globulus Interpretation K. pneumoniae NCTC 9633 12.0 8.0 8.0 (0.8) 6.0 8.0 (1.0) 8.0 (1.2) 6.0 0.8 Additive M. catarrhalis ATCC 23246 8.0 8.0 8.0 ( 1.0) 3.5 4.0 (0.8) 4.0 (0.8) 4.0 0.7 Additive E. faecalis ATCC 29212 7.0 9.0 8.0 ( 1.0) 2.5 4.0 (1.1) 4.0 (1.0) 4.0 0.9 Additive C. neoformans ATCC 90112 4.0 0.5 0.5 ( 0.6) 0.8 0.4 (0.3) 2.0 (3.3) 3.0 2.3 Indifferent S. pneumoniae ATCC 49619 8.0 1.5 2.0 (0.8) 14.0 2.0 (0.2) 4.0 (1.5) 4.0 1.2 Indifferent S. pyogenes ATCC 8668 5.3 1.8 1.5 (0.6) 1.3 2.0 (0.9) 2.0 (1.3) 2.0 1.0 Additive S. agalactiae ATCC 55618 1.7 1.5 2.0 (1.3) 1.5 1.0 (0.6) 1.0 (0.7) 2.0 1.3 Indifferent M. smegmatis (clinical) 8.0 4.0 4.0 (0.8) 4.0 4.0 (0.8) 4.0 (1.0) 4.0 0.8 Additive *Values in bold indicate noteworthy activity. Shaded area: results previously discussed in Chapter 3, 4 and 5 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. 153 The aqueous extracts of the combination of A. afra with O. asteriscoides yielded interactions against seven pathogens to be additive and one indifferent. The combination of the extracts of A. afra with E. globulus showed varied interactions with, two noteworthy synergistic (against C. neoformans and S. pneumoniae) profiles. For the combination of O. asteriscoides with E. globulus, indifferent number of varied interactions were also noted, none of which were antagonistic. The triple combination is administered as an infusion (van Wyk and Wink, 2004). The results obtained correlate with the traditional use and the advantage of the administration of the triple combination can be seen, as none of the interactions obtained against the pathogens demonstrated any adverse effects. 8.2.2 A. afra, E. globulus and L. randii in combination The botanical description, locality, medicinal uses and phytochemistry of A. afra is discussed in Chapter 3 and similarly of E. globulus and L. randii are discussed in Appendix A4 and A5. The dichloromethane: methanol extracts of A. afra, E. globulus with L. randii were evaluated using thin layer chromatography. MIC determination and ?FIC interpretation was carried out on the dichloromethane: methanol and the aqueous extracts according to the methods described in Chapter 2. Although L. randii is an aromatic plant, the essential oils were not studied as no oil was obtained during the hydrodistillation process, and thus have been excluded in the triple combination study. 8.2.2.1 Chromatographic techniques 8.2.2.1.1 Thin layer chromatography Thin layer chromatography analysis of the dichloromethane: methanol extracts of A. afra, E. globulus, and L. randii shows conjugated double bonded compounds, which are highly fluorescent at 254 nm (Figure 8.3). 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 254 nm 154 Figure 8.3 TLC plates of the dichloromethane: methanol extracts of A. afra, L. randii and E. globulus alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. At 365 nm, L. randii displayed many compounds, which showed up red and blue in colour. Under white light (Figure 8.3), L. randii showed the most coloured compound of yellow. This compound was found between Rf values 0.8 and 0.9. 1 A. afra 2 L. randii 3 E. globulus 4 A. afra with L. randii 5 A. afra with E. globulus 6 L. randii with E. globulus 7 A. afra, L. randii with E. globulus 365 nm White light Derivatized 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 155 8.2.2.2 Antimicrobial analysis 8.2.2.2.1 MIC assays and FIC determination 8.2.2.2.1.1 Dichloromethane: methanol extracts The MIC values of A. afra, L. randii and E. globulus dichloromethane: methanol extracts against the various pathogens tested are shown in Table 8.4. MIC values individually of L. randii showed noteworthy activity against M. smegmatis with a MIC value of 0.5 mg/ml. when tested against K. pneumoniae and S. pyogenes, weak inhibition was noted with mean MIC values of 2.0 mg/ml and 1.0 mg/ml respectively. Moderate inhibitory antimicrobial activity was noted against M. catarrhalis (MIC value = 1.3 mg/ml), E. faecalis (MIC value = 1.5 mg/ml) and S. pneumoniae (MIC value = 1.5 mg/ml).Very poor activity was seen against C. neoformans and S. agalactiae with MIC values of 8.0 mg/ml and 12.0 mg/ml. In the combination of the dichloromethane: methanol extracts of A. afra and L. randii mean MIC values ranged from 0.3-8.0 mg/ml. C. neoformans and M. smegmatis gave the best MIC values, with noteworthy strong inhibition, of 0.3 mg/ml and 0.5 mg/ml respectively. Against S. pyogenes, the poorest activity of the combination was noted at 8.0 mg/ml. Moderate inhibitory activity was noted against S. pneumoniae with a mean MIC value of 1.0 mg/ml. Some activity was seen against K. pneumoniae, M. catarrhalis, E. faecalis and S. agalactiae. The ?FIC values, in brackets (Table 8.4) showed synergy for C. neoformans with a ?FIC value of 0.14. Additive interactions were noted against M. catarrhalis, E. faecalis, S. agalactiae and M. smegmatis. A study was recently conducted (Eloff, 2010) testing the antimicrobial activity of the acetone extract of L. randii against four pathogens i.e. S. aureus, P. aeruginosa, E. coli and E. faecalis. Results showed good antimicrobial activities with MIC values below 0.1 mg/ml against S. aureus (0.058 mg/ml), P. aeruginosa (0.033 mg/ml) and E. faecalis (0.015 mg/ml). The MIC value against E. coli was 0.113 mg/ml. The study concluded that after toxicity and stability studies are carried out, L. randii could be promising as a commercial antibacterial agent. The combination of E. globulus and L. randii showed noteworthy inhibitory antimicrobial activity against K. pneumoniae (MIC value = 0.5 mg/ml), C. neoformans (MIC value = 0.5 mg/ml), S. pneumoniae (MIC value = 0.3 mg/ml) and M. smegmatis (MIC value = 0.5 mg/ml). 156 Table 8.4 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, L. randii, and E. globulus alone and in combination. *Values in bold indicate noteworthy activity Shaded area: results previously discussed in Chapter 3 and 6 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml)/ ?FIC value indicated in brackets where applicable ?FIC A. afra L. randii A. afra with L. randii E. globulus A. afra with E. globulus L. randii with E. globulus A. afra, L. randii with E. globulus A. afra, L. randii and E. globulus Interpretation K. pneumoniae NCTC 9633 6.3 2.0 4.0 (1.3) 3.0 2.0 (0.5) 0.5 (0.23) 0.8 0.3 Synergy M. catarrhalis ATCC 23246 4.7 1.3 2.0 (1.0) 0.01 1.1 ( 5.8) 1.4 (36.55) 0.04 1.0 Additive E. faecalis ATCC 29212 6.3 1.5 2.0 (0.8) 0.02 2.1 ( 7.6) 1.0 (25.1) 0.04 0.8 Additive C. neoformans ATCC 90112 1.2 8.0 0.3 (0.1) 0.5 0.8 (1.1) 0.5 (0.5) 0.3 0.3 Synergy S. pneumoniae ATCC 49619 0.5 1.5 1.0 (1.3) 0.2 0.3 (0.9) 0.3 (0.9) 0.3 0.8 Additive S. pyogenes ATCC 8668 1.1 1.7 8.0 (3.0) 0.6 0.3 (0.3) 1.0 (1.1) 0.8 0.8 Additive S. agalactiae ATCC 55618 3.0 12.0 4.0 (0.8) 1.0 1.0 (0.7) 2.0 (1.1) 8.0 3.8 Indifference M. smegmatis (clinical) 1.7 0.5 0.5 (0.7) 1.5 0.5 (0.3) 0.5 (0. 7) 0.5 0.5 Synergy 157 Noteworthy moderate inhibition was obtained against E. faecalis with a MIC value of 0.99 mg/ml. Some inhibition was seen against M. catarrhalis, S. pyogenes and S. agalactiae. ?FIC values for this combination gave synergistic activity against K. pneumoniae (?FIC value = 0.23) and additive interactions against C. neoformans, S. pneumoniae and M. smegmatis. Antagonism was noted against M. catarrhalis and E. faecalis. The triple combination, A. afra with E. globulus and L. randii, yielded noteworthy activity against all pathogens tested except against S. agalactiae, which gave very poor activity with an MIC value of 8.0 mg/ml. The best antimicrobial activity, and very strong inhibition, of the triple combination was against M. catarrhalis and E. faecalis with mean MIC values of 0.04 mg/ml. Against K. pneumoniae and S. pyogenes moderate inhibitory activity was noted, while against C. neoformans, S. pneumoniae and M. smegmatis, strong inhibition was evident. ?FIC values of the triple combination thus gave additive interactions against M. catarrhalis, E. faecalis, S. pneumoniae and S. pyogenes. Synergistic inhibition was obtained against K. pneumoniae, C. neoformans and M. smegmatis. Comparisons of this study with that of L. randii acetone extracts (Eloff, 2010) showed some variation. This variation could be due to the difference in extract prepared or due to different plant collection sites. For the combination of A. afra with L. randii (dichloromethane: methanol extracts) against the eight pathogens tested, four of them demonstrated additive interactions with one antagonistic (S. pyogenes) and one synergistic (C. neoformans). The interactions for the combination of A. afra with E. globulus gave three synergistic interactions against K. pneumoniae, S. pyogenes and M. smegmatis. Two pathogens displayed antagonism (M. catarrhalis and E. faecalis). The combination of L. randii with E. globulus gave one synergistic interaction against K. pneumoniae, three additive interactions and two antagonistic interactions (M. catarrhalis and E. faecalis). The combination of A. afra with L. randii and E. globulus displayed varied interactions, depending on the pathogen studied. The triple combination is better than the double combinations as no antagonism was noted and positive results (synergistic and additive interactions) were obtained. The traditional use of the triple combination is cited as being administered as an infusion. However, due to the results obtained, the use of a tincture might be a more viable option. 158 8.2.2.2.1.2 Aqueous extracts The aqueous extracts of L. randii independently, as shown in Table 8.5, proved ineffective in inhibiting E. faecalis, S. pneumoniae, and S. pyogenes at the highest concentration tested. Some activity was noted against K. pneumoniae, C. neoformans, S. agalactiae and M. smegmatis and very poor activity was obtained against M. catarrhalis. For the combination of A. afra with L. randii, no activity at the highest concentration tested was noted against S. pneumoniae and S. pyogenes. Some activity was noted against K. pneumoniae, E. faecalis, C. neoformans, S. agalactiae and M. smegmatis. Very poor activity was seen against M. catarrhalis, with a MIC value of 12.0 mg/ml. ?FIC values calculated for the combination gave additive interactions against K. pneumoniae, C. neoformans and M. smegmatis. Tentative interpretations against S. pneumoniae and S. pyogenes showed additive or antagonistic interactions present. In the combination of E. globulus with L. randii aqueous extract moderate inhibitory activity with MIC values of 1.0 mg/ml was obtained against C. neoformans and S. pneumoniae. Noteworthy strong inhibition was obtained against M. smegmatis (MIC value = 0.5 mg/ml). ?FIC values calculated showed additive interactions of E. globulus with L. randii aqueous extract against K. pneumoniae and C. neoformans. A synergistic interaction against M. smegmatis was shown with a ?FIC value = 0.2. The combination against S. agalactiae was interpreted as being antagonistic. In the combination of the three plants together i.e. A. afra, E. globulus with L. randii, very poor activity with a MIC value of 16.0 mg/ml and 8.0 mg/ml was noted against S. pneumoniae and M. catarrhalis respectively. Some activity was seen against K. pneumoniae, E. faecalis, C. neoformans, S. agalactiae and M. smegmatis. The ?FIC values calculated, showed an additive interaction against K. pneumoniae. Tentative interpretations of the interactions of the triple combination showed synergistic or additive activity against E. faecalis and additive or indifferent activity against S. pneumoniae. 159 Table 8.5 MIC and ?FIC values of the aqueous extracts of A. afra, L. randii, and E. globulus alone and in combination. Micro-organism MIC (mg/ml)/ ?FIC value indicated in brackets where applicable ?FIC A. afra L. randii A. afra with L. randii E. globulus A. afra with E. globulus L. randii with E. globulus A. afra, L. randii with E. globulus A. afra, L. randii with E. globulus Interpretation K. pneumoniae NCTC 9633 12.0 6.0 6.0 (0.8) 6.0 8.0 (1.0) 6.0 (1.0) 4.0 0.7 Additive M. catarrhalis ATCC 23246 8.0 8.0 12.0 (1.5) 3.5 4.0 (0.8) 8.0 (1.6) 8.0 1.3 Indifferent E. faecalis ATCC 29212 7.0 ?16.0 2.0 (ND1) 2.5 4.0 (1.1) 2.0 (ND1) 2.0 ND1 Synergistic/Additive2 C. neoformans ATCC 90112 4.0 6.0 4.0 (0.8) 0.8 0.4 (0.3) 1.0 (0.7) 2.0 1.1 Indifferent S. pneumoniae ATCC 49619 8.0 ?16.0 ?16.0 (ND1) 14.0 2.0 (0.2) 1.0 (ND1) 16.0 ND1 Additive/Indifferent2 S. pyogenes ATCC 8668 5.3 ?16.0 ?16.0 (ND1) 1.3 2.0 (0.9) 6.0 (ND1) ?16.0 ND1 Antagonism/Indifferent2 S. agalactiae ATCC 55618 1.7 4.0 4.0 (1.7) 1.5 1.0 (0.6) ?16.0 (ND1) 4.0 1.6 Indifferent M. smegmatis (clinical) 8.0 2.0 3.0 (0.9) 4.0 4.0 (0.8) 0.5 (0.2) 4.0 1.4 Indifferent *Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for L. randii/combination was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 and 6 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. 160 The combination of A. afra with L. randii aqueous extract displayed three additive interactions against the pathogens tested. Tentative interpretations showed additive or antagonistic interactions against S. pneumoniae and S. pyogenes respectively. Synergy was also noted against the pathogen E. faecalis. For the combination of A. afra with E. globulus, the interactions against five pathogens showed additivity and against two pathogens synergism was noted (C. neoformans and S. pneumoniae). The interactions observed for the combination of E. globulus with L. randii against one pathogen was synergistic (M. smegmatis). Tentatively interpretations against E. faecalis were synergistic or additive. Against S. pneumoniae synergy was noted and against S. pyogenes additivity or indifference was observed. The interaction against S. agalactiae tentatively displayed antagonism. The triple combination, A. afra with L. randii and E. globulus displayed an interaction against one pathogen which was additive. Tentatively, synergy or additivity was noted against E. faecalis. Traditionally infusions of the triple combination of A. afra with L. randii and E. globulus are used to treat chest infections (Watt and Breyer-Brandwijk, 1962). The results obtained correlate somewhat with the traditional use. The combination against S. pyogenes related infections warrants caution as the possibility of antagonism is evident. In comparison with the dichloromethane: methanol extracts, the results obtained in the study for the triple combination shows that although the traditional use is through an infusion, tinctures may be a better option to treat respiratory infections. 8.2.3 A. afra, Z. capense and Allium sativum in combination Garlic (Allium sativum) has been described and proven as a ?cure all? (Harris et al., 2001). The Xhosa of South Africa drink a decoction of the leaf and the bulb of garlic as a febrifuge. This is prepared with the addition of Z. capense and A. afra (Watt and Breyer-Brandwijk, 1962). Hence, as the focus of the study was combinations with A. afra, this combination was studied. Garlic has been used for centuries in different cultures and societies (Rivlin, 2001). The antimicrobial activity has been extensively investigated (Sivam et al., 1997; Phay et al., 1999; Hsieh et al., 2001; Ward et al., 2002; Benkeblia, 2004). In Nigeria, it is used to treat abdominal discomfort, diarrhoea, otitis media and respiratory tract infections (Ankri and Mirelman, 1999; Jaber and Al-Mossawi, 2007; Abubakar, 2009). 161 The botanical description, locality, medicinal uses and phytochemistry of A. sativum, A. afra (Chapter 3) and Z. capense (Chapter 7) are discussed in Appendix A2, A3 and A9 respectively. The dichloromethane: methanol extracts of A. afra, Z. capense and A. sativum were evaluated using thin layer chromatography. MIC determination and ?FIC interpretation was carried out on the, dichloromethane: methanol and the aqueous extracts according to the methods described in Chapter 2. 8.2.3.1 Chromatographic techniques 8.2.3.1.1 Thin layer chromatography A large number of compounds were seen under the different wavelengths for each individual plant sample and majority of these were seen in the combination samples. In the combination of all three it is evident that majority of the same bands that appear in the individual TLC?s are present in the combination. At Rf = 0.6, a major compound in A. sativum was found. At 365 nm, A. sativum shows pooling of the compounds at the bottom of the plate. The major green coloured compound found in Z. capense is seen in the combination with A. afra (Figure 8.4, lane 5), A. sativum (Figure 8.4, lane 6) and in the triple combination (Figure 8.4, lane 7) at Rf = 0.85. 1 A. afra 2 A. sativum 3 Z. capense 4 A. afra with A. sativum 5 A. afra with Z. capense 6 A. sativum with Z. capense 7 A. afra, A. sativum with Z. capense 254 nm 365 nm 1 2 3 4 5 6 7 1 2 3 4 5 6 7 162 Figure 8.4 TLC plates of the dichloromethane: methanol extracts of A. afra, Z. capense with A. sativum alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. The combination of A. afra, Z. capense and A. sativum shows at 254 nm, 365 nm, under white light and after derivatization, the complexity of the mixture and the presence of compounds from each individual plant in the combination. 8.2.3.2 Antimicrobial analysis 8.2.3.2.1 MIC assays and FIC determination 8.2.3.2.1.1 Dichloromethane: methanol extracts As shown in Table 8.6, A. sativum dichloromethane: methanol extracts generally showed poor to no activity at the highest concentration tested. The highest antimicrobial activity for A. sativum was against C. neoformans, which gave an MIC value of 2.0 mg/ml. The combination of A. afra with A. sativum showed moderate activity against M. catarrhalis (MIC value = 1.0 mg/ml) and S. pyogenes (MIC value = 1.5 mg/ml). Some antimicrobial activity was noted against C. neoformans, S. pneumoniae and M. smegmatis. Derivatized White light 1 2 3 4 5 6 7 1 2 3 4 5 6 7 163 Table 8.6 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, Z. capense, and A. sativum alone and in combination. *Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for A. sativum/combination was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 and 7 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml)/ ?FIC value indicated in brackets where applicable ?FIC A. afra Z. capense A. afra with Z. capense A. sativum A. afra with A. sativum Z. capense with A. sativum A. afra, Z. capense with A. sativum A. afra, Z. capense with A. sativum Interpretation K. pneumoniae NCTC 9633 6.3 1.7 2.0 (0.8) ?16.0 8.0 (ND1) 4.0 (ND1) 6.0 ND 1 Indifferent2 M. catarrhalis ATCC 23246 4.7 2.0 2.0 (0.7) ?16.0 1.0 (ND1) 8.0 (ND1) 1.8 ND1 Indifferent2 E. faecalis ATCC 29212 6.3 1.3 2.0 (0.9) ?16.0 6.0 (ND1) 12.0 (ND1) 3.0 ND1 Indifferent2 C. neoformans ATCC 90112 1.2 0.4 0.4 (0.7) 2.0 2.0 (1.3) 0.8 (1.2) 1.2 1.5 Indifferent S. pneumoniae ATCC 49619 0.5 1.5 0.3 (0.4) 8.0 2.0 (2.1) 4.0 (1.6) 1.5 1.4 Indifferent S. pyogenes ATCC 8668 1.1 2.0 1.0 (0.7) 16.0 1.5 (0.7) 4.0 (1.1) 2.0 1.0 Additive S. agalactiae ATCC 55618 3.0 4.0 4.0 (1.2) 16.0 ?16.0 (ND1) 16.0 (2.5) 10.7 2.3 Indifferent M. smegmatis (clinical) 1.7 0.8 2.0 (1.9) ?16.0 4.0 (ND1) 1.5 (ND1) 0.5 ND1 Additive/Synergy2 164 Poor to no activity was noted against K. pneumoniae, E. faecalis and S. agalactiae. ?FIC values calculated showed additive interactions against S. pyogenes and indifference against C. neoformans and S. pneumoniae. In the combination of the dichloromethane: methanol extracts of Z. capense with A. sativum, the best antimicrobial activity showing noteworthy moderate inhibition, was noted against C. neoformans with an MIC value of 0.8 mg/ml. Moderate inhibitory activity was seen against M. smegmatis with a MIC value of 1.5 mg/ml. Very poor activity was noted against K. pneumoniae, M. catarrhalis, S. pneumoniae, S. pyogenes, E. faecalis and S. agalactiae. Indifferent interactions were calculated against C. neoformans, S. pneumoniae, S. pyogenes and S. agalactiae while tentative interpretations of indifference were made against K. pneumoniae, M. catarrhalis, E. faecalis and M. smegmatis. For the triple combination i.e. A. afra, Z. capense with A. sativum dichloromethane: methanol extracts (Table 8.6), noteworthy strong inhibition was noted against M. smegmatis with a MIC value of 0.5 mg/ml. Moderate inhibition was noted against C. neoformans and S. pneumoniae. Some activity was obtained against K. pneumoniae, M. catarrhalis, E. faecalis and S. pyogenes. Very poor activity was seen against S. agalactiae. ?FIC values calculated for the triple combination gave an additive interaction against S. pyogenes. Tentative ?FIC interpretations showed indifferent interactions against K. pneumoniae, M. catarrhalis and E. faecalis and synergistic or additive interactions against M. smegmatis. The combination of A. afra with Z. capense dichloromethane: methanol extracts shows the interactions to be mostly additive, with one interaction displaying synergism (S. pneumoniae). For the combination of A. afra with A. sativum, only three interactions could be calculated, wherein two pathogens showed indifference (C. neoformans and S. pneumoniae) and one was additive (S. pyogenes). Tentative interpretations were made for the rest of the five pathogens. Two pathogens showed additive or indifferent interactions (E. faecalis and M. smegmatis), one pathogen was either indifferent or antagonistic (S. agalactiae), one was additive (K. pneumoniae) and one pathogen displayed synergy (M. catarrhalis). In the combination of Z. capense with A. sativum, mostly indifference was observed. The triple combination displayed varied interactions but mostly indifference. The results indicate that 165 the administration of a triple combination instead of any of the double combinations would have no benefit with respect to increased antimicrobial activity. 8.2.3.2.1.2 Aqueous extracts Table 8.7 shows the antimicrobial activity of the aqueous extracts of A. afra, Z. capense, and A. sativum independently and in various combinations with each other. A. sativum aqueous extract showed no activity at the highest concentration tested against K. pneumoniae, M. catarrhalis, E. faecalis, S. pneumoniae, S. agalactiae and M. smegmatis. However, against S. pyogenes, very poor activity with a MIC value of 12.0 mg/ml was obtained. Moderate inhibition was found against C. neoformans and the MIC value obtained was 1.0 mg/ml. The aqueous garlic extract was tested by Farbman et al. (1993) using the macro-dilution MIC assay. Results showed that S. pneumoniae was sensitive to a 1:2 dilution. It was ascertained that garlic had a broad spectrum of activity. Jaber and Al-Mossawi (2007) tested the water, ethanol, dichloromethane, n-hexane extracts and the raw juice of garlic against different strains of S. aureus and E. coli, Salmonella goldcoast and Klebsiella aerogenes. Disc diffusion and chequerboard assays we performed on the water extract and the raw juice only as they produced the best results in the preliminary screening. However, poor activities were noted with the MIC values when they were tested in water and when they were subjected to different pH conditions (acid or alkali). The water extracts did however, perform better that the solvent extracts even though poor activities were noted. Abubakar (2009) tested the ethanol, chloroform and the aqueous extracts of A. sativum using the well diffusion method against S. aureus, E. coli, S. pneumoniae and P. aeruginosa. Results obtained against S. pneumoniae were 21 mm for the ethanol extracts, 19 mm for the chloroform extracts and 23 mm for the aqueous extracts. The aqueous extract was more active than the solvent or organic extracts, which is congruent with Roy et al. (2006), Jaber, and Al-Mossawi (2007). 166 Table 8.7 MIC and ?FIC values of the aqueous extracts of A. afra, Z. capense, and A. sativum alone and in combination. Values in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for A. sativum/combination was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 and 7 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml)/ ?FIC value indicated in brackets where applicable ?FIC A. afra Z. capense A. afra with Z. capense A. sativum A. afra with A. sativum Z. capense with A. sativum A. afra, Z. capense with A. sativum A. afra, Z. capense with A. sativum Interpretation K. pneumoniae NCTC 9633 12.0 13.3 12.0 (1.0) ?16.0 16.0 (ND1) 8.0 (ND1) 16.0 ND1 Additive/Indifference2 M. catarrhalis ATCC 23246 8.0 16.0 8.0 (0.8) ?16.0 16.0 (ND1) 12.0 (ND1) 12.0 ND1 Indifference2 E. faecalis ATCC 29212 7.0 16.0 ?16.0 (ND1) ?16.0 ?16.0 (ND1) 12.0 (ND1) 4.0 ND1 Synergy2 C. neoformans ATCC 90112 4.0 2.0 4.0 (1.5) 1.0 1.5 (0.9) 4.0 (3.0) 1.0 0.6 Additive S. pneumoniae ATCC 49619 8.0 ?16.0 ?16.0 (ND1) ?16.0 ?16.0 (ND1) 2.0 (ND1) ?16.0 ND1 Indifference/Antagonism2 S. pyogenes ATCC 8668 5.3 ?16.0 ?16.0 (ND1) 12.0 16.0 (2.2) 4.0 (ND1) ?16.0 ND1 Indifference/Antagonism2 S. agalactiae ATCC 55618 1.7 0.4 1.3 (2.0) ?16.0 3.0 (ND1) ?16.0 (ND1) 1.0 ND1 Additive/Indifference2 M. smegmatis (clinical) 8.0 ?16.0 3.0 (ND1) ?16.0 8.0 (ND1) 1.0 (ND1) 1.0 ND1 Synergy/Additive2 167 When A. afra was combined with A. sativum aqueous extracts, MIC values were not obtained against E. faecalis and S. pneumoniae at the highest concentration tested. Very poor antimicrobial activity was seen against K. pneumoniae, M. catarrhalis, S. pyogenes and M. smegmatis. Some activity was noted against S. agalactiae with a MIC value of 3.0 mg/ml. Moderate inhibitory activity (MIC value of 1.5 mg/ml) was obtained against C. neoformans. When the ?FIC values were calculated, additive interactions were noted against C. neoformans and indifferent activity was seen against S. pyogenes. When Z. capense was combined with A. sativum, MIC values were varied from 1.0-12.0 mg/ml. Very poor activity was noted against K. pneumoniae, M. catarrhalis, and E. faecalis. Some activity was seen against C. neoformans, S. pneumoniae and S. pyogenes. The best activity (MIC value = 1.0 mg/ml) found for the combination was moderate inhibitory activity and it was obtained against M. smegmatis. ?FIC values for the combination of A. sativum with Z. capense showed an indifferent interaction against C. neoformans. For the combination of the three plants i.e. A. afra, A. sativum and Z. capense, no activity was seen against S. pneumoniae and S. pyogenes while very poor activity was noted against K. pneumoniae and M. catarrhalis. Some antimicrobial activity was noted against E. faecalis with a MIC value of 4.0 mg/ml. The best activity of the triple combination was found against C. neoformans as well as against S. agalactiae and M. smegmatis with moderate inhibitory activity (MIC values of 1.0 mg/ml). A ?FIC value of 0.6 and denoting an additive interaction were obtained for the combination of the three plants against C. neoformans. Tentative interpretations of the MIC data on the interactions of the combination showed additive or indifferent interactions against K. pneumoniae and S. agalactiae. An indifferent interaction was noted against M. catarrhalis and indifference or antagonistic interactions against S. pneumoniae and S. pyogenes was seen. Synergy was evident against E. faecalis and additivity or synergy was noted against M. smegmatis. The combination of A. afra with Z. capense (aqueous extracts) showed the interactions to be varied depending on the pathogen studied. The combination of A. afra with A. sativum showed additivity against one pathogen (C. neoformans) and an indifferent interaction against another (S. pyogenes). The combination of A. sativum with Z. capense resulted in interactions against three pathogens (S. pneumoniae, S. pyogenes and M. smegmatis) as synergy. The triple combination showed only one definite additive interaction against C. neoformans 168 therefore the triple combination could be advantages in E. faecalis related infections. However, care should be taken against S. pneumoniae and S. pyogenes as the possibility of antagonism exists. In light of these interactions, the results do not show any significant enhancement of antimicrobial activity with the triple combination. Thus, the addition of the third plant by traditional healers could possibly due to the presence of other pharmacological properties that may assist in holistic healing. These properties could contribute to the treatment of the respiratory diseases by producing anti-inflammatory, anti-histamine, decongestant, anti- pyretic or analgesic effects. 8.3 Conclusions ? The TLC results of the combination of A. afra with O. asteriscoides and E. globulus; the combination of A. afra, E. globulus and L. randii; and the combination of A. afra with Z. capense and A. sativum highlighted the complexity of the plants being tested individually and in combination. ? The MIC values of the combination of A. afra with O. asteriscoides and E. globulus essential oils showed noteworthy antimicrobial activity against S. pneumoniae, S. pyogenes and M. smegmatis. Antagonism was seen when the ?FIC values were determined against C. neoformans. Additivity was also evident against K. pneumoniae, M. catarrhalis, E. faecalis and S. agalactiae. ? The MIC values of the dichloromethane: methanol extracts of the combination of A. afra with O. asteriscoides and E. globulus showed noteworthy activity against C. neoformans, S. pneumoniae and S. agalactiae. Synergistic activity was noted when the ?FIC values were calculated against K. pneumoniae and S. agalactiae. Antagonism was noted against M. catarrhalis and E. faecalis. ? Very good antimicrobial activity was noted for the combination of A. afra with E. globulus and L. randii dichloromethane: methanol extracts with noteworthy MIC values against all the pathogens tested with the exception of S. agalactiae. The ?FIC values of the triple combination showed predominantly additive and synergistic activity against all the pathogens tested, except for the indifference noted against S. agalactiae. 169 ? The aqueous extracts of the combination of A. afra with E. globulus and L. randii showed some antimicrobial activity against K. pneumoniae, E. faecalis, C. neoformans, S. agalactiae and M. smegmatis. Additive interactions were calculated against K. pneumoniae. ? The dichloromethane: methanol extracts of the combination of A. afra with A. sativum and Z. capense showed noteworthy inhibitory activity against M. smegmatis. ? Moderate inhibition was seen in the MIC values, of the aqueous extracts of the combination of A. afra with A. sativum and Z. capense, obtained against C. neoformans, S. agalactiae and M. smegmatis. Indifference was predominant in the interactions of the combination with synergy noted against E. faecalis. Synergy or additive interactions were noted against M. smegmatis. 170 Chapter 9 Artemisia afra in combination with adjuncts 9.1Introduction An adjunct is added as a supplementary agent to an active drug and is not an essential part of the formulation (http://www.encyclopedia.com/topic/adjunct.aspx). With respect to pharmacological applications, an example would be the use of syrup (sugar) to mask the taste of paracetamol in Panado?. The addition of the green colour and flavouring is also an example of the use of an adjunct. In this case, it is added to provide flavour and is more aesthetically pleasing to children. The use of adjuncts with medicinal plants by traditional healers is a popular, yet unstudied area in phytomedicine (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; van Wyk et al., 2008). Investigation of traditional medicinal practices have revealed a number of instances where plants are mixed with milk, honey, salt, butter, and yoghurt to improve the acceptability of the medicine (Roberts, 1990; Hutchings et al., 1996; J?ger, 2003; Johnston, 2009; Thring and Weitz, 2006; van Wyk et al., 2008; van Wyk, 2008b; Liu, et al., 2009; Poonam and Singh, 2009). A combination of plants are frequently used and sometimes mixed with adjuncts before administration, the most common being honey, milk, sugar, salt, fat and brandy (Figure 9.1). Traditional preparations often require sugar or honey to make syrups (van Wyk et al., 2008). The use of these provides a sweet vehicle for the medicament, allowing palatability of the plant and thus making the patient more comfortable with swallowing it. Fat or oil are also used as vehicles, perhaps to increase viscosity for topical applications or to provide an oily vehicle for eardrops (Thring and Weitz, 2006; van Wyk, 2008b). They are readily available and the familiarity of these vehicles provides a level of comfort when being used by the patients. Sometimes milk is used instead of water when making decoctions or teas perhaps because of the availability or even because a particular plant may sometimes be more soluble in milk than water (Bhattarai et al., 2006). Brandy and vinegar are used to make tinctures (Watt and Breyer-Brandwijk, 1962). They are also commonly used when preparing poultices for wounds. 171 Figure 9.1 The various adjuncts used in traditional medicinal practices. (Roberts, 1990; Hutchings et al., 1996; J?ger, 2003; Johnston, 2009; Thring and Weitz, 2006; van Wyk et al., 2008; van Wyk, 2008b; Liu, et al., 2009). One example where the practical use of adjuncts is applied is the use of the leaf and stem of Lippia javanica as a weak tea made with either water or milk for coughs, colds and bronchial complaints (van Wyk et al., 2009; Watt and Breyer-Brandwijk, 1962). Another example is the use of A. afra with honey or sugar for coughs and colds (Roberts, 1990; Liu et al., 2009). It is also commonly used with brandy for infantile colic (Watt and Breyer-Brandwijk, 1962). These and other examples are elaborated on in Table 9.1. Aside from the practical applications of these adjuncts i.e. to aid in medicinal plant delivery, it is possible that each adjunct could have a therapeutic effect. There are many instances where these adjuncts are used independently as remedies. There are documented incidents where vinegar and honey have been used for wound care from the time of Hippocrates (Johnston, 2009). In fact, evidence suggests that vinegar on its own has therapeutic value, in the management of blood glucose, as suggested by Johnston (2009). In order to accurately assess the activity and effectiveness of the plants, it is important to take into account the Plant Honey Milk Sugar Salt Vinegar Brandy Oil/fat 172 traditional preparation of the medicine (J?ger, 2003). The addition of honey or sugar could prove to have an effect on the extent to which that plant works. Table 9.1 Various plants used in combination with adjuncts. Plant/s Adjunct Use Complaint Reference A. afra with Tetradenia riparia Salt Decoction Coughs Hutchings et al. (1996). A. afra with Warburgia salutaris Honey, brown sugar Infusion Cough Felhaber, (1997). Agathosma betulina Vinegar, brandy Soaked leaves, poultice, sprayed Wounds, sprains, contusions Thring and Weitz, (2006); van Wyk, (2008b). A. afra Honey, sugar Infusion, decoction Bronchial troubles, coughs and colds, chills, dyspepsia, loss of appetite, stomach ache, colic, croup, whooping cough, gout and as a purgative. Roberts, (1990); Liu et al. (2009). A. afra Brandy, vinegar Moistened leaf Infantile colic Watt and Breyer- Brandwijk, (1962). A. afra Brandy Tincture taken by mouth Colic Watt and Breyer- Brandwijk, (1962). A. afra Milk Suspension of ground up plant as enema Constipation and intestinal worms Watt and Breyer- Brandwijk, (1962). Ballota africana Sugar Syrup Colds Thring and Weitz, (2006). Carbobrotus edulis or C. acinaciformis or C. murii Milk Added to the squeezed juice of leaves Stomach troubles Thring and Weitz, (2006). Cissampelos capensis Vinegar, sugar Root infusion Fever van Wyk et al. (2008b). Clematis brachiata Honey Decoction Sinusitis Felhaber, (1997). Crassula ovata Milk Boil leaves Diarrhoea van Wyk, (2008b). Crassula tetragona Milk Boiled leaves Diarrhoea van Wyk, (2008b). Cynodon dactylon Fat Rubbed in skin Gout van Wyk, (2008b). Datura spp. Potassiu m nitrate Powdered Asthma van Wyk, (2008b). 173 Plant/s Adjunct Use Complaint Reference Eberlanzia spinosa Sugar Decoction of leaves Angina van Wyk et al. (2008). Elytropappus rhinocerotis Brandy, vinegar Leaves are soaked and taken, Soaked leaves on a cloth, wrapped around sore area Bladder, kidney, convulsions, diabetes, fever, headache, cough, cold, flu, stomach-ache Thring and Weitz, (2006). Euryops multifidus Fat Resins mixed with fats, applied Sores van Wyk, (2008b). Exomis microphylla/ Exomis axyroides Milk Leaf decoction Khoi remedy for epilepsy; winds, cramps and convulsions in infants van Wyk, (2008b). Galenia africana Salt Boil in water and wash wounds Wounds van Wyk et al. (2008). Galium tomentosum Sugar Infusion of the roots Remove acid in babies van Wyk et al. (2008). Gnidia polycephala Milk Drink a root infusion mixed with milk, purgative Constipation van Wyk et al. (2008). Heamanthus coccineus Vinegar Sliced bulbs Expectorant, diuretic van Wyk, (2008b). Heeria argentea Sweet oil Gum and oil mixed to make a plaster Burns. Wounds and tender nipples van Wyk, (2008b). Helichrysum litorale Fat Powdered herb, applied Ulcers van Wyk, (2008b). Lippia javanica Milk Infusion of leaf and stem Coughs, colds, bronchial troubles Watt and Breyer- Brandwijk, (1962). Melianthus comosus Vinegar Apply fresh leaves as a poultice Knee pain van Wyk et al. (2008). Pelargonium antidysenterium Milk Decoction Dysentery van Wyk, (2008b). Peliostomum origanoides Sweet oil/linsee d oil or cod liver oil Tea made from the twigs + oil Acid in babies van Wyk et al. (2008). 174 Some of these adjuncts e.g. honey and vinegar has shown independent antimicrobial activity (Wahdan, 1998; Johnston, 2009). In fact, as alternate medicine increases in popularity, many studies are aiming to verify and support the use of these traditional remedies. For a number of years the interest in the benefits of honey has increased. The antimicrobial activity of honey alone, at various concentrations, has been established. These are for bacteria commonly found to infect wounds, as well as a number of fungi (Efem et al., 1992; Molan, 1996; Malika et al., 2005; Alandejani et al., 2009). These studies further provide an interest in the possible contribution of honey and other adjuncts to the efficacy of traditional plant medicines. This study was undertaken with the aim of finding a scientific rationale for the use of adjuncts in combination with medicinal plants that are commonly used in South African traditional medicine. As the primary focus of this study, A. afra was chosen as the main plant around which adjunct studies has focussed. This plant was chosen due to its prominence as Plant/s Adjunct Use Complaint Reference Pteronia onobromoides Fat Mixed with powdered leaf Burns, sunburn, earache van Wyk, (2008b). Schinus molle Vinegar Place fresh leaves in a cloth, with vinegar and apply to the head Headache van Wyk et al. (2008). Sutherlandia microphylla Honey Drink General medicine van Wyk et al. (2008). Trichilia emetica Milk, honey Mixed with powdered roots Asthma, cough, bronchial trouble, fever, vomiting Sutovska et al. (2009). Tulbaghia alliacea milk Infusion Intestinal worms, fever, influenza high blood pressure tuberculosis van Wyk, (2008b). Tulbaghia violecea Milk Infusion Intestinal worms, fever, influenza, high blood pressure, TB van Wyk, (2008b). Tulbaghia violecea Castor oil Ear drops Earache Thring and Weitz, (2006). Tulbaghia violecea Brandy Seed-pods soaked and taken Sleeplessness, heart condition, relieves stomach pain, convulsions Thring and Weitz, (2006). Viscum capense Brandy Whole pieces soaked and taken Fever, cold symptoms Thring and Weitz, (2006). 175 one of the foremost plants used frequently in combinations with other plants as well as with adjuncts. This is shown Table 1, where A. afra was commonly found to be combined with honey, salt, brandy, and milk (Watt and Breyer-Brandwijk, 1962). 9.2 Results and discussion 9.2.1 Individual MIC values Individual MIC values of adjuncts show no antimicrobial activity against M. catarrhalis, E. faecalis, K. pneumoniae and C. neoformans. Exceptions were noted with honey where very low activity was seen against K. pneumoniae. Vinegar was also poorly active against K. pneumoniae and C. neoformans with MIC values of 16.0 mg/ml. Table 9.2 documents the individual MIC values for all adjuncts. Table 9.2 Individual mean MIC values of all adjunct samples tested. 9.2.2 Honey in combination with A. afra The different types of honey used in this study were commercially purchased orange blossom honey, wild blossom honey and blue gum honey. Sample MIC values (mg/ml) M. catarrhalis ATCC 23246 E. faecalis ATCC 29212 K. pneumoniae NCTC 9633 C. neoformans ATCC 90112 Honey ?16.0 ?16.0 8.0 ?16.0 Orange Blossom Honey (100%) ?25.0 ?25.0 ?25.0 ?25.0 Wild Blossom Honey (100%) ?25.0 ?25.0 ?25.0 ?25.0 Blue Gum Honey (100%) ?25.0 ?25.0 ?25.0 ?25.0 Salt (4%) ?10.0 ?10.0 ?10.0 ?10.0 Skim milk ?25.0 ?25.0 ?25.0 ?25.0 Full cream milk ?25.0 ?25.0 ?25.0 ?25.0 Brandy ?16.0 ?16.0 ?16.0 ?16.0 Vinegar ?16.0 ?16.0 16.0 16.0 176 The testing of different honey samples was carried out to determine if there is any differing microbial activity among differing types of honey. Studies conducted on honey samples in the past have shown that honey from different sources may display different activities (Lee et al., 2008). Honey (100%) was used as it displayed good antimicrobial activity in a previous study (Efem et al., 1992). The MIC values represented in Table 9.3 are those of the different types of honey in combination with the essential oil and extracts of A. afra. Significant values to note are those of honey at 64.0 mg/ml with the A. afra aqueous extract, which shows considerably better MIC values than that of honey alone. Against M. catarrhalis and E. faecalis, some activity was obtained with a MIC of 3.0 mg/ml. Similarly, moderate inhibition was obtained against K. pneumoniae with a MIC of 1.5 mg/ml. For the 100% blue gum honey in combination with the dichloromethane: methanol extracts of A. afra, some activity was noted against K. pneumoniae and C. neoformans. A. afra aqueous extract in combination with 100% orange blossom or wild blossom showed some activity against E. faecalis and K. pneumoniae. Moderate inhibitory antimicrobial activity (1.3 mg/ml) was noted against C. neoformans in the combination of A. afra dichloromethane: methanol extract and 100% orange blossom honey. Although MIC values can be interpreted as being favorable in the combinations tested, a better understanding of the interactions of the combinations can be sought by calculating of ?FIC values. In the testing of the use of adjuncts in combination with A. afra most of the results of the MIC values showed no end point at the concentrations tested. Thus, bar graphs have been constructed to make comparisons of the ?FIC interactions. Figure 9.2 shows the ?FIC values of the combinations against the four different micro-organisms tested. In the combination of A. afra and honey at a concentration of 64.0 mg/ml, the majority of combinations showed indifference. The combination of orange blossom honey at 64.0 mg/ml in combination with A. afra aqueous extracts against C. neoformans showed an additive interaction. 177 Table 9.3 MIC values for different types of honey in combination with A. afra samples. Sample MIC values (mg/ml) M. catarrhalis ATCC 23246 E. faecalis ATCC 29212 K. pneumoniae NCTC 9633 C. neoformans ATCC 90112 A. afra essential oil 8.0 8.0 8.0 13.0 A. afra dichloromethane: methanol extracts 4.0 4.0 2.0 4.3 A. afra aqueous extract 4.0 8.0 8.0 13.0 A. afra essential oil: honey (64.0 mg/ml) 16.0 16.0 16.0 48.0 A. afra essential oil: orange blossom honey (100%) 57.0 57.0 57.0 28.5 A. afra essential oil: wild blossom honey (100%) 57.0 57.0 57.0 14.3 A. afra essential oil: blue gum honey (100%) 57.0 57.0 57.0 14.3 A. afra dichloromethane: methanol extracts: honey (64.0 mg/ml) 8.0 8.0 8.0 22.0 A. afra dichloromethane: methanol extracts orange blossom honey (100%) 10.3 10.3 5.1 1.3 A. afra dichloromethane: methanol extracts wild blossom honey (100%) 10.3 10.3 5.1 2.6 A. afra dichloromethane: methanol extracts blue gum honey (100%) 10.3 5.1 2.6 2.5 A. afra aqueous extract: honey (64.0 mg/ml) 3.0 3.0 1.5 8.0 178 Sample MIC values (mg/ml) M. catarrhalis ATCC 23246 E. faecalis ATCC 29212 K. pneumoniae NCTC 9633 C. neoformans ATCC 90112 A. afra aqueous extract: orange blossom honey (100%) 26.0 5.1 2.6 41.0 A. afra aqueous extract: wild blossom honey (100%) 26.0 5.1 2.6 41.0 A. afra aqueous extract: blue gum honey (100%) 26.0 10.3 10.3 41.0 In the combination of A. afra essential oil and orange blossom honey at 100%, antagonism was observed. The dichloromethane: methanol extract showed synergistic activity against C. neoformans. The aqueous extracts showed additivity against K. pneumoniae, and antagonism against E. faecalis and C. neoformans. These results show a high incidence of antagonism with essential oil and aqueous extract combinations of A. afra with honey. In traditional healing practices, essential oils are seldom combined with honey. Synergism seems to be restricted to the combinations where dichloromethane: methanol extracts are combined with all three 100% honey products against C. neoformans. Results obtained support the assumption that the addition of honey is mainly for sweetening the plant decoctions thereby disguising any unpleasant taste that may be encountered by the patient. 179 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 M. ca ta rr h a li s E. fae cal is K . pn eumonia e C . n eoforman s M. ca ta rr h a li s E. fae cal is K . pn eumonia e C . n eoforman s M. cata rr ha li s E. fae cal is K . pn eumonia e C . n eoforman s M. ca ta rr h a li s E. fae cal is K . pn eumonia e C . n eoforman s A. afra and orange blossom honey (64mg/ml) A. afra and orange blossom honey (100%) A. afra and wild blossom honey (100%) A. afra and blue gum honey (100%) ? F IC v alue s Micro-organism Essential oil Dichloromethane: methanol extract Aqueous extracts Figure 9.2 The ?FIC interactions of the different types of honey in combination with A. afra. ? Antagonism Indifference Additive Synergy 180 9.2.3 Skim milk and full cream milk in combination with A. afra The MIC values obtained for the combination of A. afra together with skim milk and full cream milk are shown in Table 9.4. Both these were tested as the traditional healer uses either one depending on circumstances. MIC values in combination show poor activity. However, against M. catarrhalis, in the combination of A. afra aqueous extract and skim milk display some antimicrobial activity (MIC value of 5.1 mg/ml). Table 9.4 MIC values of A. afra in combination with milk. Figure 9.3 shows the ?FIC interaction of the combination of skim or full cream milk with A. afra essential oils, dichloromethane: methanol and aqueous extracts. The interactions with skim milk show mainly indifference. Exceptions were seen in the combination of A. afra Sample MIC values (mg/ml) M. catarrhalis ATCC 23246 E. faecalis ATCC 29212 K. pneumoniae NCTC 9633 C. neoformans ATCC 90112 A. afra essential oil 8.0 8.0 8.0 13.0 A. afra dichloromethane: methanol extracts 4.0 4.0 2.0 4.3 A. afra aqueous extract 4.0 8.0 8.0 13.0 A. afra essential oil: skim milk 28.5 57.0 28.5 14.3 A. afra essential oil: full cream 57.0 28.5 28.5 41.5 A. afra dichloromethane: methanol extracts: skim milk 20.5 20.5 20.5 20.5 A. afra dichloromethane: methanol extracts: full cream milk 20.5 20.5 10.3 20.5 A. afra aqueous extract: skim milk 5.1 20.5 41.0 30.7 A. afra aqueous extract: full cream milk 20.5 ?41.0 41.0 41.0 181 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 M . catar rhal is E. fae cal is K . pn eumonia e C . neo fo rma n s M . catar rhal is E. fae cal is K . pn eumonia e C . neo fo rma n s A. afra and skim milk A. afra and full cream milk ?F IC va lues Micro-organism Essential oil Dichloromethane: methanol extracts Aqueous extracts aqueous extract with skim milk where an additive interaction was noted against M. catarrhalis. Figure 9.3 A. afra in combination with milk. An additive interaction was also noted for the essential oil and skim milk combination against C. neoformans. Antagonism was seen against E. faecalis for the essential oil combination in both skim milk and full cream milk. Antagonism was also evident against K. pneumoniae and E. faecalis for the aqueous extract combination with skim milk and full cream milk respectively. Essential oils are not known to be administered with milk and hence the antagonism noted with the essential oils may not be significant when observing traditional practises. Milk may possibly help alter the taste of the medicine and therefore make it more acceptable to the patient to consume. For further consideration, there is the possibility that the role of milk may be more beneficial as an excipient in the formulation to improve the absorption of the plant mixture. In addition, as milk has a soothing and calming connotation for many people it may also be employed as a means to increase comfort to the gastrointestinal tract of the patient. ? Antagonism Indifference Additive Synergy 182 With the exception of one or two instances, it is also noted that the addition of milk has no negative impact on the antimicrobial activity of A. afra. 9.2.4 Vinegar and/brandy in combination with A. afra The concentration of the vinegar sample tested was 64.0 mg/ml. The MIC values obtained when vinegar was combined with A. afra essential oil showed very poor activities (Table 9.5). Table 9.5 A. afra in combination with vinegar and brandy. In combination with the dichloromethane: methanol extract, some activity was noted against M. catarrhalis with a MIC value of 4.0 mg/ml. Essential oil and aqueous extract combinations with vinegar have antagonistic interactions against the pathogens tested in this study. The ?FIC values for all pathogens and in combination with essential oil and water Sample MIC values (mg/ml) M. catarrhalis ATCC 23246 E. faecalis ATCC 29212 K. pneumoniae NCTC 9633 C. neoformans ATCC 90112 A. afra essential oil 8.0 8.0 8.0 13.0 A. afra dichloromethane: methanol extract 4.0 4.0 2.0 4.3 A. afra aqueous Extract 4.0 8.0 8.0 13.0 A. afra essential oil: brandy 48.0 48.0 48.0 24.0 A. afra essential oil: vinegar 48.0 48.0 48.0 24.0 A. afra dichloromethane: methanol extract : brandy 4.0 16.0 16.0 32.0 A. afra dichloromethane: methanol extract : vinegar 16.0 8.0 8.0 16.0 A. afra aqueous extract: brandy 32.0 32.0 32.0 4.0 A. afra aqueous extract: vinegar 32.0 32.0 16.0 8.0 183 extracts show values >4.0 (Figure 9.4). The dichloromethane: methanol extract show additive interactions against M. catarrhalis and E. faecalis. All the A. afra combinations with vinegar showed indifference against C. neoformans. One of the possible reasons for antagonistic actions could be that the main use of vinegar is in softening of the leaves for the treatment of colic. The testing of pathogens known to cause gastrointestinal distress may yield better interactive efficacies. Brandy is one of the most commonly used adjuncts to medicinal plants. It is mainly used to soften and soak plant leaves and twigs. The brandy is also commonly used in preparations for wounds (Table 9.1). The MIC values of brandy in combination with A. afra are shown in Table 9.5. Poor antimicrobial activities were noted in the essential oil, dichloromethane: methanol extract and the aqueous extract combinations against all four pathogens tested. Figure 9.4 A. afra in combination with vinegar. One exception with some activity (MIC value of 4.0 mg/ml) was obtained against C. neoformans for the aqueous extract combination of A. afra with brandy. 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 M. catarrhalis E. faecalis K. pneumoniae C. neoformans ? F IC v alue s Micro-organism Essential oil Dichloromethane: methanol extracts Aqueous extracts Antagonism Indifference Additive Synergy ? 184 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 M. catarrhalis E. faecalis K. pneumoniae C. neoformans ? F IC v alue s Micro-organism Essential oil Dichloromethane: methanol extracts Aqueous extracts Figure 9.5 Brandy in combination with A. afra. Brandy, in combination with the essential oil and water extract of A. afra show antagonistic interactions against three bacteria tested (Figure 9.5). This supports the common, modern medicinal practice of discouraging the imbibing of brandy and other alcoholic drinks with medication. The dichloromethane: methanol extract, on the other hand showed additive interactions against M. catarrhalis and C. neoformans. Indifference was noted against E. faecalis and K. pneumoniae. This provides some credibility to the most common use in the preparation of tinctures and poultices. Further studies, using other plants and bacteria specifically for wound healing are needed to explore this possibility. 9.2.5 Salt in combination with A. afra Salt is considered as an antimicrobial agent as it works by restricting bacterial growth. This is done in two ways. Either by lowering the amount of free water molecules around the bacteria e.g. in food or body tissues. The salt water creates a hypertonic environment then causing water to rush out of the bacteria by osmosis and thus killing or rupturing the cells. Moisture is essential to bacterial growth and survival and without enough water, they do not grow well. Furthermore, bacteria do not thrive or grow well in salty environments. Salt restricts microbial growth by interfering with the microbe?s enzyme activity and thus weakens its Antagonism Indifference Additive Synergy ? 185 DNA molecular structure (Parish, 2006; Shee et al., 2010). The study by Shee et al. (2010) tested the potential of salt and sugar as food preservatives. A 2.5% salt solution was used to test the preservative efficacy. Results demonstrated that the solution was able to contain the growth of bacteria tested (not specified) until the third day. The study by Milne et al. (2007) examined the effects of a 15% salt impregnated dressing for critically colonized wounds. Results showed a reduction of bacteria in critically colonized wounds in the majority of the patients following the 24 hrs after application. The MIC values obtained for the combination of salt with A. afra essential oil, dichloromethane: methanol and aqueous extract are shown in Table 9.6. The MIC values show very poor activity against all the pathogens for the essential oils, dichloromethane: methanol and aqueous extracts. The results of the interactions of A. afra and salt combinations are shown in Figure 9.6. All pathogens in combination with essential oil, aqueous extract, and dichloromethane: methanol extracts respond with indifference (?FIC values calculated were between 1.0 and 4.0). Table 9.6 MIC values of salt in combination with A. afra. Sample MIC values (mg/ml) M. catarrhalis ATCC 23246 E. faecalis ATCC 29212 K. pneumoniae NCTC 9633 C. neoformans ATCC 90112 A. afra essential oil 8.0 8.0 8.0 13.0 A. afra dichloromethane: methanol extracts 4.0 4.0 2.0 4.3 A. afra aqueous extract 4.0 8.0 8.0 13.0 A. afra essential oil: salt 21.0 21.0 18.5 21.0 A. afra dichloromethane: methanol extracts: salt 13.0 13.0 13.0 6.5 A. afra aqueous extract: salt 13.0 26.0 13.0 26.0 186 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 M. catarrhalis E. faecalis K. pneumoniae C. neoformans ? F IC v alue s Micro-organism Essential oil Dichloromethane: methanol extracts Aqueous extracts Although essential oil salt combination values are slightly lower than the other extracts it is not significantly low to be additive. The use of salt as a flavour enhancer does not in any way diminish the antimicrobial activity of the plant. Figure 9.6 The interaction of A. afra with salt. One can assume that the addition of these said adjuncts are possibly used for other purposes by traditional healers. Some of the possible uses include the use of honey to enhance taste, especially in children. Milk could be helpful in producing a possible enzymatic reaction. Salt could also be primarily added to mask unpleasant bitterness of the medicinal plant. Brandy and vinegar may be used in the making of poultices to create a cooling sensation when they evaporate or even just as a means to soften the leaf and roots of the plant. 9.2.5.1 The combination of A. afra, Tetradenia riparia and salt Salt, together with A. afra and T. riparia is used as a decoction for the treatment of coughs (Hutchings et al., 1996). The use of salt in combination with plants is also mentioned in Adams et al. (2009) where it states that salt is used in combination with the crushed leaves of Plantago lanceolata and are then placed on arthritic limbs (Bock, 1577; Matthiolus, 1590; Lonicerus, 1770). ? Antagonism Indifference Additive Synergy 187 Furthermore, it is also worth considering that as the combination of A. afra with T. riparia and salt is typically ingested, the salt content may play a role in the dissolution and/or absorption of the active components. It is known that the salt form of drugs e.g. salicylic acid shows faster dissolution and absorption rates. Salts increase dissolution rates thus increasing absorption rates. When salt encounters aqueous environments, it rapidly dissolves and takes the compounds bound to it with it in solution. This enables easier absorption (Kerns and Di, 2008). When used as an application to the skin, the salt may have a cooling effect or the rough texture may be beneficial in helping to stimulate blood flow in these areas. Thus, the administration of the combination is essential to ultimately understanding the possible benefits of these medicinal plants. The antimicrobial activity of the combination of T. riparia with A. afra and salt has not been tested previously and represents a new area for further in depth analysis into the implications of administration methods and additives to the overall activity and effectiveness of medicinal plants 9.2.5.1.1 Chromatographic techniques 9.2.5.1.1.1 Essential oil composition of T. riparia Essential oil was distilled from T. riparia, which was collected from the Walter Sisulu Botanical gardens in Johannesburg. Forty-two compounds were identified in the essential oil, representing 91.2% of the total composition (Table 9.7). The major compounds in the essential oil comprised of ?-terpinene (4.1%), ?-caryophyllene (32.4%), germacrene D (6.3%), bicyclogermacrene (4.8%), and ?-cadinol (5.4%). Table 9.7 Essential oil composition of T. riparia. RRI* Compound name Area (%) 1016 ?-Pinene 0.1 1019 ?-Thujene 0.1 1104 ?-Pinene 0.1 1117 Sabinene 0.3 1193 ?-Terpinene 1.9 1193 Limonene 0.1 1203 ? ?Phellandrene 0.1 1242 ?-Terpinene 4.1 1270 p-Cymene 1.0 1281 Terpinolene 1.1 1701 Verbenene 0.1 188 RRI*: Relative retention indices calculated against n-alkanes. Area (%) calculated from FID data. Tr = Trace; *UD: Undetermined. Major peaks: 288, 273, 245, t: tentative identification. Campbell et al. (1997) have reported on the composition of the essential oil of T. riparia from South Africa. Thirty-five compounds were identified and the main constituents found in the study were ?-terpineol (22.6%), fenchone (13.6%), ?-fenchyl alcohol (10.7%), ?- caryophyllene (7.9%) and perillyl alcohol (6.0%). A study by Omolo et al. (2004) revealed the major compound present in the essential oil of T. riparia from Kenya to be fenchone (?65%), limonene (?2.0%) and 1, 8-cineole (1.5%). RRI* Compound name Area (%) 1456 trans Sabinene hydrate 3.4 1448 ?-Copaene 0.3 1511 ?-Bourbonene 1.2 1528 ?-Gurjunene 0.5 1546 Linalool 0.2 1562 Terpinen-4-ol acetate 1.0 1586 ?-Elemene 0.2 1589 ?-Copaene 0.1 1595 ?-Caryophyllene 32.4 1627 cis-Menth-2-en-1-ol 0.7 1647 allo Aromadendrene 0.3 1674 ?-Humulene 0.9 1701 ?-Terpineol 0.7 1715 Germacrene D 6.3 1728 ?-Muurolene 0.8 1741 Bicyclogermacrene 4.8 1763 ?-Cadinene 2.1 1768 ?-Cadinene 0.4 1861 p-Cymen-9-ol 0.2 1900 Cubebol 0.1 1948 Palustrol 2.6 2009 Caryophyllene oxide 0.7 2067 Germacrene-4-D-ol 0.2 2084 Cubenol 0.5 2103 Guaiol 0.1 2141 Spathulenol 0.5 2185 T-Cadinol 1.1 2196 Eusdesmol 2.4 2201 ?-Cadinol 5.4 2327 Undetermined* 9.8 Abiatatriene (t) 2.3 Total 91.2 189 A recent study (Gazim et al., 2010) was carried out on the seasonal variation, chemical composition and analgesic and antimicrobial properties of the essential oil from the leaves of T. riparia in Brazil. The authors reported a total of 36 compounds identified from the essential oil, accounting for 95.0-99.6% of the volatile constituents. The major constituents found were 14-hydroxy-9-epi-caryophyllene (?20%), cis-muurolol-5-en-4-?-ol (?10%), ?- cardinol (?6%), ledol (?6%), calyculone (?20%), abietadiene (?10%) and fenchone (?10%). The current study showed the major compound present as ?-caryophyllene at 32.4% with an unidentified compound at 9.8%. This is different from the composition found by Campbell et al. (1997), where ?-caryophyllene was only found at 7.9% and Gazim et al. (2010) found ?- caryophyllene in small amounts only. Omolo et al. (2004) did not find any ?-caryophyllene in the essential oil at all. In fact, fenchone was the main compound in Omolo et al. (2004), but no trace was found in the essential oil of T. riparia in this study. The differences in the constituents of the essential oils of T. riparia in South Africa and Kenya, and Brazil may be due to differences in environmental factors, chemotype, nutrition of the plants and genetics (Gazim et al., 2010). 9.2.5.1.1.2 Thin layer chromatography In previous TLC assays, two major bands were found for T. riparia (Scott et al., 2004). A grey band with an Rf value of 0.36 (in a toluene: diethyl ether: 1.75 M acetic acid solvent) and 0.76 and another which was the 1,8-cineole marker at Rf value of 0.79 which was light blue in colour (Scott et al., 2004; Scott and Springfield, 2004). Similar bands were noted in the T. riparia extracts in the current study. The same three major bands were visible in Figure 9.7, at 254 nm, 365 nm and after derivatization. These bands were also visible in the combination with salt (Figure 9.7, lane 7) and in the triple combination. 190 Figure 9.7 TLC plates of the dichloromethane: methanol extracts of A. afra, Tetradenia riparia with salt alone and in combination at 254 nm, 365 nm, white light and visualized with vanillin-sulphuric acid reagent. 1 A. afra 2 T. riparia 3 Salt 4 A. afra with T. riparia 5 A. afra with salt 6 T. riparia with salt 7 A. afra, T. riparia and salt 254 nm 365 nm White light 1 2 3 4 5 6 7 1 2 3 4 5 6 7 Derivatized 1 2 3 4 5 6 7 1 2 3 4 5 6 7 191 9.2.5.1.2 Antimicrobial analysis 9.2.5.1.2.1 Essential oils In Table 9.8, the MIC values of the essential oils of T. riparia alone and in combination with either salt or A. afra or all three together. For studies on T. riparia noteworthy activity was seen against C. neoformans (MIC value = 2.0 mg/ml), S. agalactiae (MIC value = 1.5 mg/ml) and M. smegmatis (MIC value = 2.0 mg/ml). The combination of the essential oils of A. afra with T. riparia demonstrated noteworthy activity against C. neoformans, S. agalactiae and M. smegmatis. Salt showed no antimicrobial activity against K. pneumoniae, M. catarrhalis, E. faecalis, C. neoformans, S. pneumoniae and S. pyogenes at the highest concentration tested. However, some activity was noted against S. agalactiae and noteworthy activity was seen against M. smegmatis (MIC value = 0.6 mg/ml). The combination of A. afra with salt showed noteworthy activity only against M. smegmatis (MIC value of 0.6 mg/ml) was obtained. For the combination of T. riparia with salt, some antimicrobial activity was noted. Noteworthy activity was seen against M. smegmatis with a MIC value of 1.2 mg/ml. For the combination of A. afra, T. riparia and salt noteworthy antimicrobial activity was noted against C. neoformans, S. pneumoniae and M. smegmatis. The most significant activity was noted for M. smegmatis (MIC value of 0.2 mg/ml). ?FIC values calculated revealed and synergy against M. smegmatis. Tentative interpretations showed varied interactions ranging from synergism to antagonism. 9.2.5.1.2.2 Dichloromethane: methanol extracts In table 9.9 the results of the dichloromethane: methanol extracts of A. afra and T. riparia alone and in combination with salt are presented. For T. riparia independently noteworthy inhibitory activity were noted against C. neoformans, S. pneumoniae and S. pyogenes and S. agalactiae (MIC values of 0.5, 0.3, 0.4 and 0.6 mg/ml respectively). Moderate inhibitory activity was noted for the combination of A. afra with T. riparia against K. pneumoniae, M. catarrhalis, E. faecalis and M. smegmatis. Noteworthy strong inhibition was obtained against S. pneumoniae with a MIC value of 0.5 mg/ml. 192 The combination of A. afra with salt showed some activity against C. neoformans and S. agalactiae and noteworthy moderate inhibition against M. smegmatis (MIC value = 0.7 mg/ml). For the combination of T. riparia with salt, noteworthy activity for the combination was obtained against C. neoformans with a MIC value of 0.3 mg/ml. MIC values obtained for the triple combination of A. afra with T. riparia and salt showed noteworthy activity (values range between 0.1 and 0.5 mg/ml) against all the pathogens tested with the exception of E. faecalis and S. agalactiae where poor activity was noted. The ?FIC values calculated showed antagonism against S. agalactiae and additivity against M. smegmatis. Tentative interpretations of the interactions of the triple combination showed synergistic activity against K. pneumoniae and M. catarrhalis. 9.2.5.1.2.3 Aqueous extracts Table 9. 10 shows the MIC values obtained for the aqueous extracts of the combination of A. afra with T. riparia and salt against the eight pathogens tested. T. riparia aqueous extract showed moderate inhibition against E. faecalis (MIC value = 1.5 mg/ml). The triple combination of the aqueous extracts showed some activity against K. pneumoniae, M. catarrhalis, E. faecalis and C. neoformans. Noteworthy strong inhibition was evident against M. smegmatis (MIC value of 0.5 mg/ml). ?FIC values calculated showed synergy against M. smegmatis with a ?FIC value of 0.3. A previous antimicrobial study on T. riparia (Boily and van Puyvelde, 1986), an initial screening of activity, where the agar dilution method was used. The methanol extracts (80%) of the leaves, stems, root fruit, and flowers of T. riparia were tested against Bacillus subtilis, Candida albicans, M. smegmatis, P. aeruginosa, S. aureus, and Salmonella gallinarum. The leaves inhibited the growth of B. subtilis, C. albicans, M. smegmatis, and P. aeruginosa. The stems did not show any inhibitory activity. The roots inhibited only B. subtilis and S. aureus, while the fruits only inhibited the growth of C. albicans. The flowers showed good antimicrobial action by inhibiting B. subtilis, C. albicans, M. smegmatis, and S. aureus. 193 Table 9.8 MIC and ?FIC values of the essential oils A. afra, T. riparia, and salt alone and in combination. *Values given in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for salt/combination was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml)/)?FIC value indicated in brackets where applicable ?FIC A. afra T. riparia A. afra with T. riparia Salt (2%) A. afra with salt T. riparia with salt A. afra, T. riparia with salt A. afra, T. riparia with salt Interpretation K. pneumoniae NCTC 9633 8.0 7.0 8.0 (1.1) ?5.0 9.3 (ND1) 9.3 (ND1) 12.3 ND1 Indifference/Antagonism2 M. catarrhalis ATCC 23246 8.7 8.0 8.0 (1.0) ?5.0 9.3 (ND1) 9.3 (ND1) 12.3 ND1 Indifference/Antagonism2 E. faecalis ATCC 29212 8.7 32.0 8.0 (0.6) ?5.0 18.5 (ND1) 3.8 (ND1) 12.3 ND1 Indifferent2 C. neoformans ATCC 90112 3.8 2.0 1.0 (0.4) ?5.0 2.3 (ND 1) 2.3 (ND1) 1.4 ND 1 Indifferent2 S. pneumoniae ATCC 49619 3.3 8.0 4.0 (0.9) ?5.0 18.5 (ND1) 9.3 (ND1) 1.4 ND 1 Synergistic/Additive2 S. pyogenes ATCC 8668 1.7 6.0 6.0 (2.3) ?5.0 9.3 (ND 1) 9.3 (ND1) 4.3 ND1 Indifferent2 S. agalactiae ATCC 55618 4.8 1.5 2.0 (0.9) 3.8 9.3 (2.2) 2.3 (1.1) 5.8 2.2 Indifferent M. smegmatis (clinical) 1.5 2.0 1.0 (0.6) 0.6 0.6 (0.7) 1.2 (1.2) 0.2 0.2 Synergy 194 Table 9.9 MIC and ?FIC values of the dichloromethane: methanol extracts of A. afra, T. riparia, and salt alone and in combination. *Values given in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for salt/combination was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml))/?FIC value indicated in brackets where applicable ?FIC A. afra T. riparia A. afra with T. riparia Salt (2%) A. afra with salt T. riparia with salt A. afra, T. riparia and salt A. afra, T. riparia and salt Interpretation K. pneumoniae NCTC 9633 6.3 1.3 1.0 (0.5) ?5.0 10.5 (ND1) ?10.5 (ND1) 0.1 ND 1 Synergy2 M. catarrhalis ATCC 23246 4.7 4.0 1.0 (0.2) ?5.0 10.5 (ND1) ?10.5 (ND1) 0.1 ND 1 Synergy2 E. faecalis ATCC 29212 6.3 1.3 1.5 (0.7) ?5.0 ?10.5 (ND1) ?10.5 (ND1) 6.2 ND1 Indifference/Antagonism 2 C. neoformans ATCC 90112 1.2 0.5 2.0 (2.8) ?5.0 2.6 (ND 1) 0.3 (ND1) 0.4 ND 1 Indifference/Additive2 S. pneumoniae ATCC 49619 0.5 0.3 0.5 (1.3) ?5.0 ?10.5 (ND 1) ?10.5 (ND1) 0.4 ND 1 Indifferent2 S. pyogenes ATCC 8668 1.1 0.4 8.0 (3.6) ?5.0 ?10.5 (ND 1) ?10.5 (ND1) 0.8 ND 1 Indifference/Additive2 S. agalactiae ATCC 55618 3.0 0.6 4.0 (4.0) 3.8 4.9 (1.5) 2.6 (2.5) 6.2 4.7 Antagonistic M. smegmatis (clinical) 1.7 1.0 1.0 (0.8) 0.6 0.7 (0.7) 1.3 (1.7) 0.5 0.6 Additive 195 Table 9.10 MIC and ?FIC values of the aqueous extracts A. afra, T. riparia, and salt alone and in combination. *Values given in bold indicate noteworthy activity. ND1 No ?FIC value could be calculated as no MIC end point for salt/combination was obtained at the highest concentration tested. 2 Tentative interpretation according to MIC data. Shaded area: results previously discussed in Chapter 3 but included here for reference purposes. Controls excluded for the sake of brevity. Please refer to Chapter 3 for further detail regarding control values. Micro-organism MIC (mg/ml))/?FIC value indicated in brackets where applicable ?FIC A. afra T. riparia A. afra with T. riparia Salt (2%) A. afra with salt T. riparia with salt A. afra, T. riparia and salt A. afra, T. riparia and salt Interpretation K. pneumoniae NCTC 9633 12.0 ?16.0 2.0 (ND1) ?5.0 5.3 (ND1) 2.6 (ND1) 3.1 ND1 Synergy2 M. catarrhalis ATCC 23246 8.0 2.5 ?16.0 (ND1) ?5.0 10.5 (ND1) 5.3 (ND1) 6.2 ND1 Indifferent2 E. faecalis ATCC 29212 7.0 1.5 ?16.0 (ND1) ?5.0 10.5 (ND1) 2.6 (ND1) 6.2 ND1 Indifferent2 C. neoformans ATCC 90112 4.0 2.0 4.0 (1.5) ?5.0 2.6 (ND1) 2.6 (ND1) 1.6 ND1 Indifference/Additive2 S. pneumoniae ATCC 49619 8.0 ?16.0 ?16.0 (ND1) ?5.0 ?10.5 (ND1) ?10.5 (ND1) ?10.5 ND1 Indifference/Antagonism2 S. pyogenes ATCC 8668 5.3 ?16.0 8.0 (ND1) ?5.0 ?10.5 (ND1) 10.5 (ND1) ?10.5 ND1 Indifference/Antagonism2 S. agalactiae ATCC 55618 1.7 ?16.0 ?16.0 (ND1) 3.8 ?10.5 (ND1) ?10.5 (ND1) ?10.5 ND1 Indifferent2 M. smegmatis (clinical) 8.0 6.0 2.0 (0.3) 1.3 2.6 (2.4) 9.8 (8.9) 0.5 0.3 Synergy 196 In 1995, Vlietinck et al. tested the antimicrobial activity of the methanol extracts of T. riparia where the best activity was obtained against a yeast. This is in partial congruence with the current study where good results were obtained against the yeast, C. neoformans. Later, McGaw et al. (2000) tested the antimicrobial activity of the methanol, hexane, and aqueous extracts, of T. riparia using the disc diffusion method. No activity, against the pathogens was noted using the discs thus further microplate MIC determination was not carried out. Comparisons with the current study are difficult as the method of antimicrobial testing differs. A recent study (Gazim et al., 2010) tested the antimicrobial activity of the essential oil of T. riparia using the disc diffusion method followed by the measurement of the MIC values using the microdilution MIC method. The most sensitive micro-organisms were S. aureus, B. subtilis, and C. albicans with the largest inhibition zones and the lowest MIC values. These antimicrobial activities obtained of T. riparia were highest in the Summer, then Autumn, Winter and the lowest in Spring. MIC values obtained against S. aureus ranged from 15.6- 31.2 ?g/ml, against B. subtilis MIC values obtained were between 7.8-15.6 ?g/ml and against C. albicans MIC values were from 31.2-62.4 ?g/ml. Good activity was also seen against other organisms tested i.e. E. faecalis, P. aeruginosa, E. coli, K. pneumoniae and S. enterica with MIC values of 62.5, 125.0 and 250 ?g/ml respectively. In the current study the most significant activity was noted for the essential oil of T. riparia against S. agalactiae with a MIC value of 1.5 mg/ml. The active principles of T. riparia which display significant antimicrobial properties are the 8(14), 15-sandaracopimaradiene-7?, 18-diol (De Kimpe et al., 1982; van Puyvelde, 1983; Boily and van Puyvelde, 1986; van Puyvelde et al., 1986). Variation between the results previously obtained could be due to the different localities of the T. riparia collected. Boily and van Puyvelde (1986) and Vleitinck et al. (1995) tested T. riparia collected from Rwanda and Gazim et al. (2010) tested T. riparia oil from Brazil. McGaw et al. (2000) tested the extracts of T. riparia, which was collected from South Africa. The T. riparia collected in this study was also from South Africa and the results obtained were moderate to poor. Thus this correlates with the findings of the antimicrobial activity that was obtained for the South African collections 197 9.3 Conclusions ? The combination of the dichloromethane; methanol extracts A. afra with orange blossom honey (100%), wild blossom honey (100%) and blue gum honey (100%) all showed synergy against C. neoformans. ? The essential oils in combination with orange blossom, wild blossom and blue gum honey are antagonistic against all the pathogens tested with the exception of the combination of A. afra with wild blossom honey (100%) and blue gum honey (100%) against C. neoformans. ? Additivity is seen for A. afra essential oil with milk (skim and or full cream) against C. neoformans, M. catarrhalis and K. pneumoniae. ? The dichloromethane: methanol extracts of A. afra are additive in combination with vinegar against E. faecalis and K. pneumoniae. ? The dichloromethane; methanol extracts of A. afra with brandy show additivity against M. catarrhalis and C. neoformans. The essential oils and aqueous extracts in combination with brandy were antagonistic against M. catarrhalis, E. faecalis and K. pneumoniae. ? Salt in combination with A. afra displays no significant interaction. Salt in the combination of A. afra with T. riparia could therefore be incorporated into the preparation to augment the taste of these two unpalatable plants in one remedy. ? The addition of an adjunct to A. afra by traditional healers is most likely due to other factors like enhancing taste or preventing bitterness. ? The results obtained from the triple combination of the essential oils i.e. A. afra, T. riparia, and salt, showed the noteworthy and very strong inhibition against M. smegmatis with a MIC value of 0.2 mg/ml. ?FIC values obtained showed synergy against M. smegmatis and antagonism against S. agalactiae. ? The dichloromethane: methanol extracts of the combination of A. afra, Tetradenia riparia, and salt showed good activity with noteworthy inhibition against all the pathogens tested except against E. faecalis and S. agalactiae. The best activity as noted against K. pneumoniae and M. catarrhalis with MIC values of 0.1 mg/ml. ?FIC values showed antagonism against S. agalactiae. ? The aqueous extracts of the combination of A. afra, Tetradenia riparia and salt showed noteworthy activity against M. smegmatis with a MIC value of 0.5 mg/ml. 198 Chapter 10 Summary and general conclusions Traditional healers often use combinations of plants or adjuncts with A. afra to treat common respiratory complaints. This study was conducted with the aim of validating the traditional use of these combinations from an antimicrobial perspective, in order to provide valuable insight into the rationale for the use of these herbal combinations. This research has provided a direction for further studies into the more complex uses of plant combinations, particularly with respect to the principle plant A. afra. An in-depth literature review was conducted and combinations of plants commonly used by traditional healers to treat respiratory infections were identified (Table 1.1). These were elaborated on and explored in Chapter 1. Five double combinations and four triple combinations were selected for further investigation. All aromatic plants where essential oil could be obtained were analysed by GC-MS and the detailed compositional analysis is provided with the corresponding plants in the thesis and monographs. A summary of the main compounds present the six plants essential oils evaluated is given in Table 10.1. Table 10.1 Summary of the major chemical constituents found from all plant essential oils in this study. Essential oil Major compounds % of major compound A. betulina isomenthone 31.4 limonene 18.9 menthone 10.0 diosphenol 8.1 pseudodiosphenol 7.5 A. afra camphor 41.0 1,8-cineole 17.0 borneol 8.6 ?-thujone 6.0 artemisia ketone 6.4 camphene 4.0 199 The TLC analysis of the plant essential oils, dichloromethane: methanol and aqueous extracts showed good separation of the compounds and the complexity of the plants individually and in combination were illustrated. MIC values for the essential oils and extracts that produced the highest activity are shown in Table 10.2. The most significant activity noted for plants studied independently was that from E. globulus dichloromethane: methanol extracts against M. catarrhalis with a MIC value of 0.01 mg/ml. From the antimicrobial interactions observed in Table 10.3, the most synergistic combinations in a 1:1 ratio is observed when A. afra is combined with O. asteriscoides (dichloromethane: methanol extracts) against S. pyogenes, when A. afra is combined with E. globulus (aqueous extracts) against S. pneumoniae, when L. randii is combined with E. globulus (dichloromethane: methanol extracts) against K. pneumoniae and when the aqueous extracts are combined against M. smegmatis in the combination of A. afra with T. riparia (dichloromethane: methanol extracts) against M. catarrhalis, all with an ?FIC value of 0.2. Essential oil Major compounds % of major compound E. globulus 1,8-cineole 63.0 ?-pinene 16.7 limonene 3.6 ?-cymene 2.7 L. javanica linalool 70.7 caryophyllene oxide 6.9 trans-linalool oxide 4.5 cis-linalool oxide 4.0 ?-cymene 3.4 1,8-cineole 2.3 O. asteriscoides 1,8-cineole 59.0 camphor 8.2 ?-terpineol 7.2 T. riparia ?-caryophyllene 32.4 germacrene D 6.3 ?-cardinol 5.4 bicyclogermacrene 4.8 ?-terpinene 4.1 200 Table 10.2 Summary of the most significant antimicrobial activity of the plant samples tested independently. Plant Plant preparation Micro-organism MIC value (mg/ml) A. afra Essential oil M. smegmatis (clinical) 1.5 Dichloromethane: methanol extract S. pneumoniae (ATCC 49619) 0.5 L. javanica Essential oil S. pyogenes (ATCC 8668) 1.4 Dichloromethane: methanol extract S. pneumoniae (ATCC 49619) 0.4 S. pyogenes (ATCC 8668) Aqueous extract S. agalactiae (ATCC 55618) 0.5 O. asteriscoides Essential oil S. pneumoniae (ATCC 49619) 1.0 Dichloromethane: methanol extract C. neoformans (ATCC 90112) 0.3 Aqueous extract C. neoformans (ATCC 90112) 0.5 A. betulina Essential oil S. pyogenes (ATCC 8668) 1.0 E. globulus Essential oil C. neoformans (ATCC 90112) 0.6 Dichloromethane: methanol extract M. catarrhalis (ATCC 23246) 0.01 Aqueous extract C. neoformans (ATCC 90112) 0.8 L. randii Dichloromethane: methanol extract M. smegmatis (clinical) 0.5 Z. capense Dichloromethane: methanol extract C. neoformans (ATCC 90112) 0.4 Aqueous extract S. agalactiae (ATCC 55618) 0.4 T. riparia Essential oil S. agalactiae (ATCC 55618) 1.5 Dichloromethane: methanol extract S. pneumoniae (ATCC 49619) 0.3 The triple combination of A. afra with T. riparia (essential oil) with salt was also synergistic (?FIC value = 0.2) against M. smegmatis. The combination of O. asteriscoides and E. 201 globulus (essential oils) against E. faecalis and the combination of A. afra with L. randii (dichloromethane: methanol extracts) against C. neoformans showed synergy with ?FIC values of 0.1. Various ratios within the isobologram interpretations demonstrated synergy and the most significant was that for the combination of A. afra with E. globulus (aqueous extract) against C. neoformans wherein all ratios tested were synergistic. Some antagonism was found and this could be important as these ratios could be highlighted as inadvisable for traditional healing practises. The interaction that was most antagonistic in a 1:1 ratio of the combination of O. asteriscoides with E. globulus (dichloromethane: methanol extracts) against M. catarrhalis where a ?FIC value of 77.3 was obtained. The isobologram studies demonstrated the highest incidence of antagonism was the combination of A. afra with E. globulus (dichloromethane: methanol extracts) against M. catarrhalis wherein five of the nine ratios were antagonistic. Table 10.3 Summary of the most significant interactions present in the plant combinations tested. Plant combination Plant part/product Micro-organism ?FIC value/isobologram ratios Interaction A. afra with L. javanica Dichloromethane: methanol extracts S. agalactiae (ATCC 55618) ?FIC value = 0.4 Synergy M. catarrhalis (ATCC 23246) Isobologram (ratios 2:8 and 1:9) Synergy E. faecalis (ATCC 29212) Isobologram (ratio 1:9) Synergy A. afra with O. asteriscoides Dichloromethane: methanol extracts S. pneumoniae (ATCC 49619) ?FIC value = 0.3 Synergy S. pyogenes (ATCC 8668) ?FIC value = 0.2 Synergy M. smegmatis (clinical) ?FIC value = 0.3 Synergy Aqueous extract M. catarrhalis (ATCC 23246) Isobologram (ratios 4:6, 3:7, 2:8 and 1:9) Synergy K. pneumoniae (NCTC 9633) Isobologram (ratio 8:2) Synergy 202 Plant combination Plant part/product Micro-organism ?FIC value/isobologram ratios Interaction E. faecalis (ATCC 29212) Isobologram (ratio 9:1) Synergy C. neoformans (ATCC 90112) Isobologram (ratios 9:1, 8:2, 6:4, 4:6, 3:7, 2:8 and 1:9) Synergy Essential oil C. neoformans (ATCC 90112) Isobologram (ratio 8:2) Synergy A. afra with A. betulina Dichloromethane: methanol extracts M. catarrhalis (ATCC 23246) ?FIC value = 0.3 Synergy Isobologram (ratios 3:7, 4:6 and 1:1) Synergy A. afra with E. globulus Dichloromethane: methanol extracts K. pneumoniae (NCTC 9633) ?FIC value = 0.5 Synergy S. pyogenes (ATCC 8668) ?FIC value = 0.3 Synergy M. smegmatis (clinical) Synergy M. catarrhalis (ATCC 23246) ?FIC value = 5.8 Antagonism E. faecalis (ATCC 29212) ?FIC value = 7.6 Antagonism Aqueous extract C. neoformans (ATCC 90112) ?FIC value = 0.3 Synergy S. pneumoniae (ATCC 49619) FIC value = 0.2 Synergy Dichloromethane: methanol extracts M. catarrhalis (ATCC 23246) Isobologram (ratios 8:2, 7:3, 6:4, 1:1 and 4:6) Antagonism K. pneumoniae (NCTC 9633) Isobologram (ratios 4:6 and 1:1) Synergy E. faecalis (ATCC 29212) Isobologram (ratios 6:4 and 1:1) Antagonism Aqueous extract M. catarrhalis (ATCC 23246) Isobologram (all ratios except 1:1) Synergy C. neoformans (ATCC 90112) Isobologram (all ratios) Synergy O. asteriscoides with E. globulus Essential oil E. faecalis (ATCC 29212) ?FIC value = 0.1 Synergy Dichloromethane: methanol extracts M. catarrhalis (ATCC 23246) ?FIC value = 77.3 Antagonism E. faecalis (ATCC 29212) ?FIC value = 34.0 Antagonism 203 Plant combination Plant part/product Micro-organism ?FIC value/isobologram ratios Interaction S. agalactiae (ATCC 55618) ?FIC value = 0.4 Synergy A. afra, O. asteriscoides with E. globulus Essential oil C. neoformans (ATCC 90112) ?FIC value = 6.5 Antagonism Dichloromethane: methanol extracts K. pneumoniae (NCTC 9633) ?FIC value = 0.5 Synergy S. agalactiae (ATCC 55618) ?FIC value = 0.4 Synergy M. catarrhalis (ATCC 23246) ?FIC value = 57.6 Antagonism E. faecalis (ATCC 29212) ?FIC value =42.9 Antagonism A. afra with L. randii Dichloromethane: methanol extracts C. neoformans (ATCC 90112) ?FIC value = 0.1 Synergy S. pyogenes (ATCC 8668) ?FIC value = 6.0 Antagonism L. randii with E. globulus Dichloromethane: methanol extracts K. pneumoniae (NCTC 9633) ?FIC value = 0.2 Synergy M. catarrhalis (ATCC 23246) ?FIC value =36.6 Antagonism E. faecalis (ATCC 29212) ?FIC value = 25.1 Antagonism Aqueous extract M. smegmatis (clinical) ?FIC value = 0.2 Synergy A. afra, L. randii with E. globulus Dichloromethane: methanol extracts K. pneumoniae (NCTC 9633) ?FIC value = 0.3 Synergy C. neoformans (ATCC 90112) ?FIC value = 0.3 Synergy M. smegmatis (clinical) ?FIC value = 0.5 Synergy A. afra with Z. capense Dichloromethane: methanol extracts S. pneumoniae (ATCC 49619) ?FIC value = 0.4 Synergy E. faecalis (ATCC 29212) Isobologram (ratio 6:4) Synergy C. neoformans (ATCC 90112) Isobologram (ratio 7:3) Synergy A. afra with T. riparia Essential oil C. neoformans (ATCC 90112) ?FIC value = 0.4 Synergy Dichloromethane: methanol extracts K. pneumoniae (NCTC 9633) ?FIC value = 0.5 Synergy 204 Plant combination Plant part/product Micro-organism ?FIC value/isobologram ratios Interaction M. catarrhalis (ATCC 23246) ?FIC value = 0.2 Synergy S. pyogenes (ATCC 8668) ?FIC value = 13.6 Antagonism S. agalactiae (ATCC 55618) ?FIC value = 4.0 Antagonism Aqueous extract M. smegmatis (clinical) ?FIC value = 0.3 Synergy T. riparia with salt Aqueous extract M. smegmatis (clinical) ?FIC value = 8.9 Antagonism A. afra, T. riparia with salt Essential oil M. smegmatis (clinical) ?FIC value = 0.2 Synergy Dichloromethane: methanol extracts S. agalactiae (ATCC 55618) ?FIC value = 4.7 Antagonism Aqueous extract M. smegmatis (clinical) ?FIC value = 0.3 Synergy Table 10.4 demonstrates a summary of the main results attained when A. afra was combined with adjuncts such as honey, milk, vinegar and brandy. The best combinational activity from the adjuncts tested in this study was obtained from the combination of A. afra (dichloromethane: methanol extracts) with orange blossom honey (100%), with wild blossom honey (100%) and blue gum honey (100%) against C. neoformans. All three interactions were synergistic. The highest antagonism noted were in the combinations of A. afra with orange blossom honey (100%), A. afra with vinegar and A. afra with brandy against M. catarrhalis, E. faecalis, K. pneumoniae and C. neoformans. Thus, care should be taken when combining these adjuncts with A. afra. Table 10.4 Summary of the adjunct interactions with A. afra. Adjunct in combination with A. afra Plant part/product Micro-organism Interaction * A. afra with orange blossom honey (64.0 mg/ml) Aqueous extract C. neoformans (ATCC 90112) Additive A. afra with orange blossom honey (100%) Essential oil M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism 205 Adjunct in combination with A. afra Plant part/product Micro-organism Interaction * K. pneumoniae (NCTC 9633) Antagonism C. neoformans (ATCC 90112) Antagonism Dichloromethane: methanol extracts C. neoformans (ATCC 90112) Synergy Aqueous extract E. faecalis (ATCC 29212) Antagonism C. neoformans (ATCC 90112) Antagonism K. pneumoniae (NCTC 9633) Additive A. afra with wild blossom honey (100%) Essential oil M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Antagonism Dichloromethane: methanol extracts C. neoformans (ATCC 90112) Synergy Aqueous extract E. faecalis (ATCC 29212) Antagonism C. neoformans (ATCC 90112) Antagonism K. pneumoniae (NCTC 9633) Additive A. afra with blue gum honey (100%) Essential oil M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Antagonism Dichloromethane: methanol extracts K. pneumoniae (NCTC 9633) Additive C. neoformans (ATCC 90112) Synergy Aqueous extract E. faecalis (ATCC 29212) Antagonism C. neoformans (ATCC 90112) Antagonism 206 Adjunct in combination with A. afra Plant part/product Micro-organism Interaction * A. afra with skim milk Essential oil E. faecalis (ATCC 29212) Antagonism C. neoformans (ATCC 90112) Additive Aqueous extract M. catarrhalis (ATCC 23246) Additive K. pneumoniae (NCTC 9633) Antagonism A. afra with full cream milk Essential oil E. faecalis (ATCC 29212) Antagonism Aqueous extract E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Additive A. afra with vinegar Essential oil M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Antagonism Dichloromethane: methanol extracts E. faecalis (ATCC 29212) Additive K. pneumoniae (NCTC 9633) Additive Aqueous extract M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Antagonism A. afra with brandy Essential oil M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Antagonism Dichloromethane: methanol extracts M. catarrhalis (ATCC 23246) Additive C. neoformans (ATCC 90112) Additive 207 Adjunct in combination with A. afra Plant part/product Micro-organism Interaction * Aqueous extract M. catarrhalis (ATCC 23246) Antagonism E. faecalis (ATCC 29212) Antagonism K. pneumoniae (NCTC 9633) Antagonism *Interactions based on data from bar graphs in Chapter 9. The purpose of this study was to validate the traditional use of A. afra combinations for the treatment of respiratory tract infections. This outcome has been achieved as many of the plant combinations have demonstrated that the essential oils have the most additivity in their combinations and thus the delivery form of inhalation may ensure that these infections are dealt with at the site of the microbial colonisation. Further in vivo studies are needed to confirm this. The results obtained from the essential oils mostly support the common use of these plants in combination as an inhalant to treat respiratory conditions. This use dates back to the origins of time when smoke or steam from a burning or boiling plant was inhaled (Mohagheghzadeh et al., 2006). Conventional medicine has also employed this method by inhaling drugs into the lungs using a nebulizer or metered dose inhalers for the treatment of respiratory illnesses. The dichloromethane: methanol extracts also showed some outstanding activity. However, some antagonism was also noted. Care should be exercised when administering tinctures as antagonism is possible. The aqueous extracts, surprisingly, showed good activity in some combinations. This corresponds with the traditional use of these combinations in the form of decoctions and infusions. The overall outcome of this study is that a valid and scientific basis for the use of these plants in combination has been established and this could prove to pave the way for future studies in the fight against bacterial respiratory infections. The study has produced positive and encouraging results that largely supports the traditional use of combinations and hopefully set the precedent for future research in this area. 208 Recommendations for future studies Further testing of the use of adjuncts in combination with A. afra (Table 10.4) could prove helpful in the development of cost-effective and efficacious treatments for primary healthcare. These adjuncts may prove their value not only as pharmaceutical additives, but may prove to have a pharmacological benefit. Some of these benefits may be an enhanced activity at receptor sites to achieve an amplified result. They may also serve to improve the dissolution of the drug, thereby increasing the absorption of the active component. Studying these effects could be beneficial in the future. Time-kill assays to determine the exact interactive effect of the combinations over time should be carried out. Toxicity studies should be undertaken to determine if toxicity is reduced or enhanced when plants are combined. The anti-oxidant, anti-inflammatory, decongestant, expectorant, antihistamine and antitussive properties should also be investigated, as plant combination selection may not only be associated with improved antimicrobial activity but also due to other pharmacological properties. There is the possibility that two plants are selected with the intention to address infection (one plant) and another related symptom for example inflammation. Thus by combining plants, two pharmacological effects are achieved within one preparation. Studies conducted should relate the synergistic interactions encountered to specific modes of action. 209 References Abubakar, E. M., 2009. Efficacy of crude extracts of garlic (Allium sativum linn.) against nosocomial Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniea and Pseudomonas aeruginosa. 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Phytochemistry, 17(10), 1795-1797. http://www.britannica.com/EBchecked/topic/325884/Kwena http://www.britannica.com/EBchecked/topic/607965/Tswana http://www.britannica.com/EBchecked/topic/363321/Manyika http://www.encyclopedia.com/topic/adjunct.aspx http://www.botanical.com/botanical/mgmh/e/eucaly14.html http://www.google.co.za/search?hl=en&biw=1280&bih=683&q=Artemisia+afra&as_q=com bination&btnG=Search%C2%A0within%C2%A0results http://www.hptlc.us/v/products/application/ats4.html http://www.camag.com/v/products/development/adc2.html http://www.camag.com/v/products/assemblies/herbal_kits5.html http://www.camag.com/v/products/derivatation/derivatization.html 248 Appendix A Monographs of plant species investigated A1Agathosma betulina (P.J. Berguis) Pillans Common names of Agathosma betulina (P.J. Berguis) Pillans are: boegoe (Afrikaans); buchu (English, Khoi; French, Italy); round leaf buchu; short buchu; ibuchu (Xhosa); Bucco (Germany) (van Wyk and Wink, 2004; Moolla, 2006; van Wyk et al., 2009). A1.1 Botanical description of A. betulina Agathosma betulina (Rutaceae) is a round leafed perennial shrub, with woody branches, of up to 2 meters in height. The leaves are small, with a rounded apex, which curves backwards, and are rich in conspicuous oil glands along the margins and lower surfaces. Thus, they are highly aromatic with a rich gold colour oil. The oil has a strong, sweetish peppermint like odour (Watt and Breyer-Brandwijk, 1962; Moolla and Viljoen, 2008). Flowers (Figure A1.1) are born at the ends, throughout August, September and October (Sigmond, 1838). They are small and star shaped in white or purple (Moolla, 2006; van Wyk et al., 2009). Figure A1.1 A. betulina flowers (A.M. Viljoen). Figure A1.2 Botanical distribution of A. betulina (SANBI). 249 A1.2 Geographical locality of A. betulina A. betulina is found in the sandy mountainous regions of the Cape of South Africa (Figure A1.2), specifically the Western Cape Province (Spreeth, 1976; Goldblatt and Manning, 2000; van Wyk and Gericke, 2000; Moolla, 2006; Moolla et al., 2007; van Wyk et al., 2009). A1.3 Medicinal uses of A. betulina A. betulina is said to be remedy for ?almost every disease which afflicts man? (Watt and Breyer-Brandwijk, 1962). Buchu brandy (boegoebrandewyn) which comprises of buchu leaves infused in brandy is a preparation made by the natives of the Cape. This preparation is considered a supreme remedy for all chronic diseases as well as for acute diseases of the stomach and bladder (Sigmond, 1838; Watt and Breyer-Brandwijk, 1962; Simpson, 1998; Viljoen et al., 2006a). It is also used as an antibiotic for protection against infections (Forbes, 1986). The leaves are sometimes chewed, or mixed in brandy to make a tincture to relieve stomach complaints or as a stomachic tonic (Gentry, 1961; Watt and Breyer-Brandwijk, 1962; van Wyk and Wink, 2004; van Wyk et al., 2009). Buchu brandy is widely known and used as a diuretic and for the treatment of kidney and urinary tract diseases (Watt and Breyer- Brandwijk, 1962; Gentry, 1961; Moolla, 2006; van Wyk et al., 2009). Buchu leaves soaked in vinegar (buchu vinegar or boegoe-asyn) is used to wash and clean wounds (Moolla, 2006; van Wyk et al., 2009). Buchu is also used to treat the symptoms of rheumatism as well as gout (Simpson, 1998; Moolla, 2006; Viljoen et al., 2006a; van Wyk et al., 2009; Vermaak et al., 2009). The essential oil of buchu is known to have therapeutic properties such as alleviating fever, coughs, colds and flu (Simpson, 1998; Viljoen et al., 2006a). A1.4 Chemical constituents of A. betulina The essential oil of buchu has been studied (Gildmeister and Treibs, 1959; Fluck et al., 1961; Guenther, 1964; Kaiser et al., 1975; Blommaert and Bartel, 1976; Posthumus et al., 1996). These studies have attributed to its flavour to 8-mercapto-p-mentha-3-one. Two monoterpene thiols were identified to be responsible for the odour (Fluck et al., 1961; Gentry, 1961; Watt and Breyer-Brandwijk, 1962; Klein and Rojahn, 1967; Lamparsky and Schudel, 1971; Kaiser et al., 1975; Simpson, 1998). Kaiser et al. (1975) isolated and identified 120 compounds in the commercial essential oil of buchu. The major compounds found were buchu camphor/diosphenol (15-30%), menthone and isomenthone (50-60%), limonene (?17%), pulegone and isopulegone (?7%). Viljoen et 250 al. (2006a) performed a study on the chemical composition of the commercially (Afriplex, South Africa) available A. betulina essential oil. They found the major constituents of buchu oil to be limonene (23.7%), pulegone (8.4%), menthone (29.2%) and isomenthone (14.2%). Non-volatile major compounds in buchu leaf extract were identified as hesperidin and diosman (El-Shafae and El-Domiaty, 2001). 251 A2 Allium sativum L. Allium sativum is also commonly known as garlic; ail blanc (French), Knoblauch (German), aglio (Italian), ajo (Spanish) and Lasan (Indian) (Ahmad and Beg, 2001; van Wyk and Wink, 2004). Allium species are said to be among the first cultivated crops in the world and its use as a spice and a medicinal plant dates back to antiquity (Sato and Miyata, 2000; Benkeblia, 2004; Rivlin, 2006). A2.1 Botanical description of A. sativum Allium sativum L. (Alliaceae) is a perennial shrub. It has fleshy slightly greyish leaves with rounded flower heads as well as numerous small bulbs, which are grouped and surrounded by a white papery sheath (Figure A2.1) (van Wyk and Wink, 2004). Figure A2.1 bulbules of A. sativum. A2.2 Geographical locality of A. sativum Garlic is reported to have come from the Middle East or central Asia (van Wyk and Wink, 2004). However, the exact origin of garlic is unknown and thus only the cultivated form is commonly used. A2.3 Medicinal uses of A. sativum It has been used as a medicinal plant for over 4000 years and is still employed in folk medicine all over the world for the treatment of a variety of diseases (Block, 1985; Ali et al., 2000; Corzo-Mart?nez et al., 2007). The oldest literature recorded, on the use of garlic as a medicine and a condiment is from the Sumerians is dated at 2600-2100 BC (Harris et al., 2001). Garlic, in its various therapeutic forms i.e. powder, extracts or essential oils have been used in traditional medicine for various ailments by the Chinese, Egyptians, Greek, Indians, Koreans, Romans and the Africans (Stoll and Seebeck, 1951; Lucas, 1966; Cha, 1978; 252 Agarwal, 1996; Ankri and Mirelman, 1999; Jaber and Al-Mossawi, 2007; Abubakar, 2009). In Africa, garlic is used to treat abdominal discomfort, diarrhoea, otitis media, and respiratory tract infections (Ankri and Mirelman, 1999; Jaber and Al-Mossawi, 2007; Abubakar, 2009). The main or primary medicinal use of garlic is as a supportive dietary treatment of hyperlipidaemia (van Wyk et al., 2009). It is also used in the treatment and prevention of the symptoms associated with the common cold (Amagase, 2006; van Wyk et al., 2009). Garlic possesses antibacterial, antifungal, antiviral, antiprotozoal, anti-oxidant, anticarcinogenic and antimutagenic, antithrombotic and anti-arthritic effects (Sato and Miyata, 1999; Ali et al., 2000; Harris et al., 2001; Corzo-Mart?nez et al., 2007; Jaber and Al-Mossawi, 2007). Additionally, it has and is being used for tuberculosis, heart problems, headaches, bites, intestinal worms, and tumours (Watt and Breyer-Brandwijk, 1962; Block, 1985; Ali et al., 2000; Corzo-Mart?nez et al., 2007). A2.4 Chemical constituents of A. sativum Garlic contains a high amount of non-volatile organo-sulpur compounds, the primary one present is S-alk(en)yl-L-cysteine sulphoxides, such as alliin and ?-glutamylcysteines. Alliin is the main compound present in intact garlic which is degraded to allicin which is most often the main compound isolated in processed garlic materials (Ahmad and Beg, 2001; van Wyk and Wink, 2004). The main biological functions of garlic are due to the presence of their high organo-sulphur compounds i.e. e alliin (Augusti & Mathew, 1974; Amagase, 2006; Corzo- Mart?nez et al., 2007). 253 A3 Artemisia afra Jacq. ex Willd. The common names of A. afra include African wormwood (English); umhlonyane, umhlonyane omcane (Xhosa, Zulu); lengana (Sotho, Tswana); als, alsem, wildeals (Afrikaans); armoise d?Afrique (French) and Afrikanischer Wermut (German) (Hutchings et al., 1996; van Wyk and Wink, 2004; van Wyk et al., 2009). A3.1 Botanical description of A. afra A. afra (Asteraceae) is an erect multi-stemmed perennial shrub. It grows up to two metres in height and it is a highly aromatic plant. It is bitter tasting and has a yellow-green volatile oil (von Koenen, 2001). It has feathery leaves (Figure A3.1) which are finely divided and have a greyish-green colour (van Wyk et al., 2009). In winter, the branches die, but rapidly regenerate from the base, and it blossoms from January to June (Hilliard, 1977; Liu et al., 2009; van Wyk et al., 2009). The flowers, which are borne along the branch ends, are pale yellowish and inconspicuous (Figure A3.2) (Hilliard, 1977; van Wyk et al., 2009). Figure A3.1 Aerial leaves of A. afra (S.F. van Vuuren) with single feathery leaf of A. afra (inset) (A.M. Viljoen). Figure A3.2 Pale yellowish flowers of A. afra (A.M. Viljoen). A3.2 Geographical locality of A. afra A. afra is a very common species and is indigenous to the mountainous regions in southern Africa (Figure A3.3) i.e. South Africa, Namibia, and Zimbabwe (Graven et al., 1992). It grows in the northern provinces of Gauteng and Limpopo along the eastern parts of South Africa as well as Swaziland and Lesotho extending up to the Western Cape of South Africa (Hilliard, 1977; Liu et al., 2009; van Wyk et al., 2009). It has an extensive distribution 254 northwards into tropical east Africa going as far north as Ethiopia (Gurib-Fakim, 2007; van Wyk et al., 2009). Figure A3.3 Geographical distribution of A. afra in South Africa (SANBI). A3.3 Medicinal uses of A. afra A. afra is typically given as root or leaf infusions, which are drunk or used as a gargle. Leaf decoctions (with sugar or honey) and an alcohol extract are taken or the steam is inhaled. This is achieved by covering the container as well as the head with a towel (making a tent) thus blocking escape of the steam and then inhaling (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; von Koenen, 2001; Thring and Weitz, 2006; van Wyk et al., 2009). It has a wide spectrum of uses such as in the treatment of fever, chills, whooping cough, loss of appetite, headache, earache, skin ulcerations, intestinal worms, colic, indigestion, flatulence, constipation, gout, rheumatism, malaria and bladder and kidney disorders (Watt and Breyer- Brandwijk, 1962; von Koenen, 2001; van Wyk and Wink, 2004; Thring and Weitz, 2006; van Wyk et al., 2009). It is also employed to bring out the rash in measles (von Koenen, 2001). A. afra is used regularly to treat respiratory problems, specifically both upper and lower respiratory tract infections (von Koenen, 2001). Among them are coughs, colds, inflammation of the throat, bronchitis, blocked nose, pneumonia and influenza (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996; van Wyk et al., 2009). 255 A3.4 Chemical constituents of A. afra Volatile as well as non-volatile constituents are found in A. afra. Non-volatile constituents from a pentane extract have revealed cerylcerotinate, nonacosane, friedeline and ?- and ?- amyrin (Silbernsgel et al., 1990; Graven et al., 1992) as well as surface flavonoids (methyl ethers of luteolin) (Wollenweber et al., 1989; van Wyk, 2008). Ascaridole, sesquiterpene lactone guaianolides, scopolectin and isofraxidin have also been identified (Jakupovic et al., 1988; Chagonda et al., 1997; Chagonda et al., 1999). Graven et al. (1992) confirms the results on the main components of the volatile oil of A. afra from Zimbabwe by Priprzek et al. (1982) and Graven et al (1986) i.e. ?-thujone (52.5%), ?- thujone (13.1%), 1,8-cineole (13.0%) and camphor (6.6%). The study on the oil from Ethiopia by Worku and Rubiolo (1996) revealed the major compound to be artemisyl acetate (24.4-32.1%). In the Kenyan oil, 1,8-cineole was the major component at 63.37% (Mwangi et al., 1995) and ?-thujone (52.5-54.2%) was the main component in the South African oil as analyzed by Lawrence (1989) and Libbey and Sturtz (1989). In the study by Chagonda et al. (1999), which studied the essential oil composition of cultivated A. afra, two chemotypes were identified i.e. cultivated from Harare and Murehwa. In the former chemotype, artemisia ketone (32.1-34.8%), camphor (21.8-24.4%), and 1,8-cineole (10.9-16.9%) were the dominant compounds. The latter sample contained 1,8-cineole (23.5-28.7%), camphor (20.2- 21.3%) and borneol (14.2-17.0%) as the major compounds. The results of this study differed completely with the studies by Graven et al. (1992), Lawrence (1989) and Libbey and Sturtz (1989) which reported ?-thujone as a major constituent of the essential oil of A. afra. These results are congruent with the studies by Moody et al. (1994) and Garnero (1977) (Chagonda et al., 1999). Mangena and Muyima (1999) studied the essential oil of A. afra from Fort Hare in South Africa and found major constituents to be ?-thujone (78.68%), ?-thujone (13.3%) and 1,8-cineole (8.19%). Once again, results differed from that of Graven et al., (1992). In 2001, Burits et al. analyzed the essential oil of A. afra from Addis Ababa, Ethiopia using GC-MS. The major compounds found were camphor (26.8%), bornyl acetate (3.8%), 4- terpineol (3.6%) and chamazulene (3.2%). Muyima et al. (2002) found camphor (15.6%), 1, 8-cineole (13.6%) and ?-thujanone (52.6%) to be the major compounds in the oil isolated from Fort Hare in South Africa. Viljoen et al. (2006b) studied the geographical variation by, GC-MS, of A. afra essential oil by comparing four natural plant populations i.e. Setibang (Lesotho), Giant?s Castle (KwaZulu Natal), QwaQwa (Free State) and Kliprviersberg 256 (Gauteng). Different plants within a population were also collected. Differences in major compounds were noted e.g. 1,8-cineole which was present in high concentrations (50%) in the Gauteng samples was low (2%) in the Lesotho population. Inter-population differences also occurred wherein ?-thujone was a major compound (77.7%) in plant sample C from Setibang and was not present at all in plant sample A of the same population. Thus it is evident that the volatile oil of A. afra is remarkably variable in the major and minor oil compounds (Graven et al., 1992; Changonda et al., 1999; Dube, 2006; Viljoen et al., 2006b; Liu et al., 2009), with a large amount of monoterpenes and sesquiterpenes present (van Wyk and Wink, 2004). This variability is possibly due to the following factors: geographical variation; different plant parts used; drying methods; variation within the natural population (Liu et al., 2009) 257 A4 Eucalyptus globulus Labill. Common names of Eucalyptus globulus Labill. are Eucalyptus (French); Blue gum; Tasmanian blue gum; true blue gum; impiskayihlangulwa, umdlavusa, umdlebe (Zulu); bloekom (Afrikaans); Eukalyptus (German); eucalipto (Italian) and eucalypto (Spanish) (Hutchings et al., 1996; van Wyk and Gericke, 2000; van Wyk and Wink, 2004; Suitor et al., 2009). A4.1 Botanical description of E. globulus E. globulus (Myrtaceae) is a very large tree (Figure A4.1). It has a characteristic shedding bark. The leaves are very glaucous, long and dimorphic as well as broad with solitary large white flowers (Figure A4.2) (Horn et al., 1964; van Wyk and Wink, 2004). Figure A4.1 Large tree of E. globulus (S.F. van Vuuren). Figure A4.2 Leaves and flowers of E. globulus (S.F. van Vuuren). A4.2 Geographical locality of E. globulus E. globulus is indigenous to Australia (Jordan et al., 1993; Dutkowski and Potts, 1999; van Wyk and Wink, 2004) and Tasmania. It is most widely cultivated in subtropical and Mediterranean regions (Takahashi et al., 2004). E. globulus was introduced into Europe, North and South Africa, California and the non-tropical districts of South America by Baron Ferdinand von M?ller, a German botanist and explorer who made the qualities known all over the world (http://www.botanical.com/botanical/mgmh/e/eucaly14.html). 258 A4.3 Medicinal uses of E. globulus Among the uses for E. globulus is as a spray to repel vermin. A poultice of E. globulus is used to draw out an abscess. The root is said to be a purgative. The leaf is used as a febrifuge and a remedy for leprosy. Leaf juice is used as a tonic and antiperiodic as well as an antiseptic (Watt and Breyer-Brandwijk, 1962). The essential oil is applied externally for the relief of rheumatism and minor skin ailments (van Wyk and Wink, 2004) The main use of E. globulus in South Africa and elsewhere is as a remedy for respiratory complaints (such as colds and fever), and is used as an ingredient in nasal drops (Watt and Breyer-Brandwijk, 1962; Felhaber, 1997). The vapour from boiling the leaves is inhaled as a respiratory antiseptic as well as for diphtheria and croup. An infusion is drunk using a bunch of slightly boiled leaves in water or pouring boiling water over bruised leaves. This is used for the treatment of influenza. Inflammation of the lungs, chest pain and difficulty in breathing is treated by boiling a few leaves and then inhaling the vapours, which can induce sleep as well (Smith, 1895; Watt and Breyer-Brandwijk, 1962; van Wyk and Wink, 2004). It is also reported by Caceres et al. (1991) to be used in the treatment of asthma, bronchitis, sore throat and chest tuberculosis, whooping cough and sinusitis. E. globulus is used for the treatment of coughs by acting as an expectorant (Mej?a, 1927; McVaugh, 1963; Morton, 1981; Alvarez, 1987; Orellana, 1987 in Caceres et al., 1991). In a study conducted by Cacreres et al. (1991), E. globulus was proven effective in inhibiting the growth of S. aureus and S. pyogenes. A4.4 Chemical constituents of E. globulus E. globulus has been one of the most studied of Eucalyptus species and the essential oil has been well characterized (Singh et al., 2000; Batista-Pereira et al., 2006; Ghalem and Mohamed, 2008a). The main constituent that was found was 1,8-cineole (70-80%) (Stecher et al., 1968; van Wyk and Wink, 2004). Other compounds include ?-pinene, phellandrene, terpineol, citronellal, geranyl acetate, eudesmol, eudesmyl acetate, piperitone, ?-cymene and limonene, camphene, ?-pinene, linalool, globulol and volatile aldehydes (isovaleric) were found (Stecher et al., 1968; Cimanga et al., 2002; van Wyk and Wink, 2004). Tohidpour et al. (2010) found eucalyptol, spathulenol, and ?-pinene as the major constituents of E. globulus. 259 Major non-volatile compounds isolated from the ethanol extract of the leaves of E. globulus was identified as containing ?-diletones, such as 16-hydroxy-18-tritriacontanone and 4- hydroxy-tritriacontane-16,18-dione (wax constituents), ellagic acid and its related compounds (Amakura et al., 2009). Other constituents include gallic acid, quercetin 3-O-?-D- glucuronide, globuluside, cryptmeriodol, 4-epi-cryptmeridiol, 3?,13?-dihydroxyurs-11-en- 28-oic acid, ?-eudesmol and macrocarpal I (Sakai et al., 2005). Amakura et al. (2009) identified the main constituents as gallic acid, oenothein, ellagic acid, quercetin 3-O-?-D- glucuronide, kaempferol 3-O-?-D-glucuronide and chlorogenic acid. Hasegawa et al. (2008) isolated two monoterpene conjugates of gallic acid, globulusin A and eucaglobulin from the hot water extract of E. globulus. 260 A5 Leonotis. randii S. Moore. Leonotis microphylla is a synonym of Leonotis ocymifolia (Burm.f) Iwaarson var. schinzii (G?rke) according to Welman in Arnold and de Wet, 2002. Leonotis randii S. Moore. is the synonym used by Klopper et al., (2006). Common names include wild dagga (English); Knopdagga, wilde-dagga, Klipdagga (Afrikaans) which translates to ?rock marijuana? (Vos, 1995) and semomonane (Watt and Breyer-Brandwijk, 1962). A5.1 Botanical description of L. randii Leonotis microphylla (Figure A5.1) is a perennial shrub (0.5-1m high) with densely covered stems, branches and leaves with long and short hispid hairs (Brown et al., 1912). It is a short plant displaying long flowering stems. Flowering of L. randii as shown in Figure A5.1 dominates in the summer rainfall season between October and February (Vos, 1995). Figure A5.1 Flower of L. randii (S.F.van Vuuren). Figure A5.2 Distribution map of L. randii (SANBI). A5.2 Geographical locality of L. randii Its recorded distribution as shown in Figure A5.2 is in Gauteng (Transvaal), Free State (Orange Free State), and Botswana (Vos, 1995). It grows primarily in the Kalahari region of Gauteng (Transvaal), as shown in Figure A5.2, in the Jeppestown Ridges of Johannesburg, Meintjies Kop in Pretoria and in Heidelberg (Skan, 1910; Brown et al., 1912). Its habitat is restricted to bankenveld, sourveld, mixed bushveld, rocky outcrops and slopes (Vos, 1995). 261 A5.3 Medicinal uses of L. randii The Africans use an infusion of Leonotis microphylla to apply to painful spots on the skin and haemorrhoids. The European and the Sotho people living in South Africa drink the infusion for the relief of digestive disturbances with fever as well as for chest affections. The Kwena, a branch of the Western Sotho people and the Tswana, Western Sotho people of South Africa drink a decoction of the plant in large doses for coughs and colds. The Manyika, people from the eastern part of Zimbabwe and across the border in Mozambique; a sub-tribe of the Shona, people in South Africa burn the flower of L. microphylla with porcupine quills and then inhale the smoke to stop nose bleeds (Watt and Breyer-Brandewijk, 1962; http://www.britannica.com/EBchecked/topic/325884/Kwena; http://www.britannica.com/EBchecked/topic/607965/Tswana; http://www.britannica.com/EBchecked/topic/363321/Manyika). A5.4 Chemical constituents of L. randii According to Vos (1995), 18 compounds were identified. The essential oil comprised of: caryophyllene 37%; germacrene 13%; eugenol 7%; phellandrene 3%; 1,8-cineole 2% and an unknown compound at retention time of 34.45 mins. 262 A6 Lippia javanica (Burm.f) Spreng Synonyms of Lippia javanica are Lantana galpiniana Pearson, Lippia asperifolia Rich or Verbena javanica Burm. f. (Hutchings et al., 1996). Common names are fever tea, lemon bush (English); musukudu and bokhukhwane (Tswana); inzinziniba (Xhosa); umsuzwane, umswazi (Zulu); mumara (Shona); koors (tee) bossie, lemoenbossie, maagbossie, beukebos (Afrikaans); musudzungwane (Tshivenda) and lufisu (Nyakyusa) (Hutchings et al., 1996; Ngassapa et al., 2003; Manenzhe et al., 2004; Samie et al., 2005; van Wyk et al., 2009). A6.1 Botanical description of L. javanica L. javanica (Verbenaceae) is an erect woody shrub growing up to two meters in height. It has hairy leaves that have conspicuous veins (van Wyk et al., 2009). The leaf is described as being highly aromatic (van Wyk et al., 2009), having the odour of vanilla or mint (Watt and Breyer-Brandwijk, 1962), as well as having a strong lemon smell. Small yellowish white flowers (Figure A6.1) are produced in dense rounded heads (van Wyk et al., 2009). Figure A6.1 Leaves of L. javanica with small yellowish white flowers (A.M. Viljoen.) Figure 6.2 Geographical distribution of L. javanica in South Africa (SANBI). A6.2 Geographical locality of L. javanica L. javanica occurs over large parts of northern South Africa and it extends northward into tropical Africa (van Wyk et al., 2009) (Figure A6.2). Predominantly, it is found in the North- eastern regions of South Africa and extends into Botswana, Zimbabwe, Malawi and Swaziland (Chagonda et al., 1993; Manenzhe et al., 2004). 263 A6.3 Medicinal uses of L. javanica L. javanica is predominantly used as an infusion for traditional medicine. Weak infusions are reportedly used as a caffeine-free tea substitute in some communities in Botswana (Watt and Breyer-Brandwijk, 1962; van Wyk et al., 2009; Shikanga et al., 2010; Manenzhe et al., 2004). Strong infusions are used topically to treat scabies and lice (van Wyk et al., 2009), it is also used as a calmative, anticolic, anticatarrh and as an emetic (Muchuweti et al., 2006). Pascual et al. (2001) documents L. javanica as being an analgesic, antipyretic, anti-inflammatory as well as being an antispasmodic and has antimicrobial and larvicidal/repellent properties (Mwangi et al., 1992; Hutchings and van Staden, 1994). It is also used to disinfect anthrax- infected meat (Watt and Breyer-Brandwijk, 1962). The Zulu speaking people from South Africa drink an infusion for the treatment of gangrenous rectitis and use the plant to treat measles, urticaria and other rashes (Watt and Breyer-Brandwijk, 1962; van Wyk et al., 2009. The cooled decoction is applied as a lotion to treat skin disorders such as rashes, stings and bites (van Wyk et al., 2009, Muchuweti et al., 2006). It is also stuffed in the nose as a remedy to stop nasal haemorrhage and colds, blackwater fever, malaria (Samie et al., 2005; Ngassapa et al., 2003), dysenteries e.g. diarrhoea (Samie et al., 2005), tapeworm infestations (Ngassapa et al., 2003), abdominal pains (Ngassapa et al., 2003) and other diseases (Watt and Breyer- Brandwijk, 1962). It is also used as a malaria remedy in South West Africa (Watt and Breyer- Brandwijk, 1962). The Kwena and the Tswane people of South Africa use a decoction for the treatment of coughs and colds. Furthermore, the smoke from the burning plant is inhaled for respiratory conditions e.g. asthma and chronic cough (Samie et al., 2005; Muchuweti et al., 2006). The leaf boiled is in water and is used as a Swati (native people from South Africa and Swaziland) remedy for influenza and colds as well as a cough remedy by the Shangana (natives of Mozambique, and South Africa) (Watt and Breyer-Brandwijk, 1962). L. javanica leaves and stems are used by the Xhosa, natives of South Africa, as a weak infusion in milk or water for the treatment of coughs, colds and bronchial troubles, shortness of breath and chest complaints (Watt and Breyer-Brandwijk, 1962; Pascual et al., 2001). L. javanica is used traditionally to treat coughs, colds, bronchitis and various chest ailments. Hot leaf infusions are taken with milk or water. 264 A6.4 Chemical constituents of L. javanica Non-volatile compounds Iridoid glucosides, theveside-NA and theviridoside, were isolated from L. javanica by Rimpler and Sauerbier (1986). The toxic triterpenoid, Icterogenin was isolated and reported by Watt and Breyer-Brandwijk (1962) and cited in Hutchings and van Staden (1994), Pascual et al. (2001), Buckingham (2006) and Mujovo et al. (2008). Phenolic secondary metabolites have been recently studied by Olivier et al. (2010). Verbascoside and isoverbascoside, isolated from the aerial parts of L. javanica, are sugar moieties and thus render them water-soluble. They are well known for their biological activities (Herbert et al., 1991; Pieroni et al., 2000; Pennacchio, 2005) thus it said that the two compounds are probably linked to the medicinal uses of L. javanica. Traditional healers use the infusions of L. javanica in most cases, and thus extraction of these active constituents is most likely (Olivier et al., 2010). The chemical composition of L. javanica essential oil has been thoroughly studied previously (Mwangi et al., 1992; Velasco-Negueruela et al., 1993; Hutchings and van Staden, 1994; Terblanche and Kornelius 1996; Ngassapa et al., 2003; Manenzhe et al., 2004; Viljoen et al., 2005). Neidlein and Staehle (1974) found caryophyllene, linalool and ?-cymene as major components of L. javanica essential oil. Later on Mwangi et al. (1991) reported the presence of myrcenone, myrcene, (E) - and (Z) - tagetenone as the main characteristic components of the same. Terblanche and Kornelius (1996) in their review reported (Z)-tagetenone (24.9- 39.9%), (E)-tagetenone(11.4-20.6%), myrcene (4.7-8.3%), myrcenone (20.9-49.7%) as the main constituents. ?-caryophyllene was also present (0.1-2.0%) (Fujita, 1965; Mwangi et al., 1991; Velasco-Negueruela et al., 1993). Samples of L. javanica essential oil from Tanzania were also analyzed by Balira (1986) and Ngassapa et al. (2003). Balira (1986) analyzed two samples, one from Kimbiji in Dares Salam (A), and the other from a southern highlands region in Tanzania Iringa (B). Sample A contained myrcenone in high amounts i.e. 52.2%. Sample B also contained myrcenone but at 9.6%. In the study by Ngassapa et al. (2003), two samples (A and B) were also chosen, but from the same location and they possessed different odours. Sample A contains limonene (9.6%), germacrene-D (9.0%), camphor (7.2%) and linalool (5.4%). Sample B had citral (geranial (21.5%) and neral (13.7%)) and limonene (11.3%). From this it is evident the two populations could be different chemotypes. 265 Manenzhe et al. (2004) sampled L. javanica essential oil in South Africa and found a major compound piperitone (74.4%), with other compounds at lower concentrations such as limonene (1.6%, linalool (1.8%), ?-isopropenylanisole (9.7%), ?-terpineol (0.8%), Z- Ocimenone (3.9%) and ?-caryophyllene (1.0%). It was reported that the essential oil is highly variable, both quantitatively and qualitatively with variations present even in the same location (Chagonda et al., 2000). In 2005, Viljoen et al. performed a study on the geographical variation of L. javanica essential oil. Five natural populations each having 16 samples were collected i.e. Swaziland, Nelspruit, Warmbaths, Long Tom Pass and Fairlands. Five different chemotypes were identified within the different populations. They were a myrcenone rich type (36-62%), a carvone rich type (61-73%), a piperitenone rich type (32- 48%), an ipsenone rich type (42-61%) and a linalool rich type (>65%). 266 A7 Osmitopsis asteriscoides (L.) Cass. Common names of O. asteriscoides include bellis, bels (e), belskruie (Afrikaans), and Mountain daisy (Bremer, 1972; Smith, 1966; van Wyk et al., 2009). A7.1 Botanical description of O. asteriscoides O. asteriscoides (Asteraceae) is a vigorous branched glabrous shrub growing up to two meters in height (Bremer, 1972). It is strongly camphor scented and has bare stems with its leaves crowded at the branch ends (van Wyk et al., 2009). The leaves are oblong and glandular displaying a large number of small glands on the surface (van Wyk et al., 2009). Due to these glands, this plant produces a high volume of essential oil. White flowers (Figure A7.1) bloom between January ? December (whole year) (Bremer, 1972), and their heads are attractive and grouped on branch tips (van Wyk et al., 2009). Figure A7.1 O. asteriscoides leaves and flowers (A.M. Viljoen). Figure A7.2 Geographical distribution of O. asteriscoides (SANBI). A7.2 Geographical locality of O. asteriscoides It is restricted to the Western Cape Province (Figure A7.2), where it thrives in open and moist ground but not in the shadow of trees (Bremer, 1972; Marloth, 1908). It then forms small ?forests? and is said to be found in swampy places on the summit of Table Mountain. A7.3 Medicinal uses of O. asteriscoides O. asteriscoides is a traditional Cape Dutch remedy for the treatment of many ailments (van Wyk et al., 2009). Brandy tinctures (Spiritus bellidis) are used for coughs and hoarseness and the dried plant extract is applied externally for inflammation. A poultice of the leaf is applied 267 to cuts and swellings. It is very popular in the treatment of stomach troubles such as colic. Included in the spectrum of medicinal uses is the use for fever, influenza, body aches and pains, and as an embrocation in paralysis (Watt and Breyer-Brandwijk, 1962). According to Watt and Breyer-Brandewijk (1962), it is reported to cause sweating. Additionally, a tincture or an infusion is applied as a remedy for rheumatism (van Wyk et al., 2009; Viljoen et al., 2003; Watt and Breyer-Brandewijk, 1962). A7.4 Chemical constituents of O. asteriscoides The essential oil of O. asteriscoides is rich in 1, 8-cineole (eucalyptol) and camphor (van Wyk et al., 2009). It has been previously studied by Bohlmann and Zdero, (1974); Bohlman et al. (1985) and Viljoen et al. (2003). O. asteriscoides has been reported to have a high amount of non- volatile sesquitepene lactones including osmitopsin isolated from the leaves (Viljoen et al., 2003; McGaw et al., 2008). 268 A8 Tetradenia riparia (Hochst.) Codd The common names of Tetradenia riparia include ginger bush (English); watersalie (Afrikaans); iboza and ibozane (Zulu) and umuravumba (Rwandanese) (Hutchings et al., 1996; van Wyk et al., 2009). Synonyms include Iboza bainesii N.E. Br., Iboza galpinii N.E. Br., Iboza riparia (Hochst.) N.E. Br., Moschosma riparia N.E. Br., and Moschosma riparium Hochst. (Hutchings et al., 1996). A8.1 Botanical description of T. riparia T. riparia (Hochst.) Codd belongs to the family Lamiaceae. It is a multi-branched shrub or a small tree (1-3 meters in height). The leaves and stems contain glandular hairs (Figure A8.1) with the leaves glandular on both surfaces (van Wyk et al., 2009). Male and female flowers (Figure A8.2), with colours ranging from white to mauve, are born on different plants. The female flower spikes are densely flowered and longer (up to 80mm long) compared to the male spikes (20m long) (Codd, 1985; van Wyk et al., 2009). Figure A8.1 Glandular leaves of T. riparia (S.F. van Vuuren). Figure A8.2 Flowers of T. riparia (H. de Wet). 269 A8.2 Geographical distribution of T. riparia T. riparia is commonly found in the eastern parts of South Africa (Figure A8.3), on dry rocky slopes, wooded hillsides or on a stream bank in relatively frost-free areas (Codd, 1985; van Wyk et al., 2009). It extends into Namibia and Angola and then north into east tropical Africa and Ethiopia (Codd, 1985; van Wyk et al., 2009). Figure A8.3 Botanical distribution of T. riparia (SANBI). 270 A8.3 Medicinal uses of T. riparia T. riparia leaf infusions and decoctions are used for respiratory diseases such as coughs, colds, sore throat, and influenza as well as other ailments such as mouth ulcers, stomach- ache, diarrhoea, haemoptysis; boils, mumps fever, malaria, swollen legs (Watt and Breyer- Brandwijk, 1962; Hutchings, 1989; Pujol, 1990; Githinji and Kokwaro, 1993; Hutchings et al., 1996; van Wyk et al., 2009). Crushed leaves of T. riparia are also inhaled for the treatment of headaches (Hutchings and van Staden, 1994). A8.4 Chemical constituents of T. riparia The major essential oil components found in T. riparia were ?-terpineol, fenchone, ?- caryophyllene, and ?-fenchyl alcohol (Campbell et al., 1997; Scott et al., 2004; van Wyk et al., 2009). Secondary chemical studies on T. riparia have revealed the presence of diterpenes which are ibozol and 7 ?-hydroxyroyleanone; the diterpenediol i.e. 8(14),15- sandaracopimaradiene-7?,18-diol which possesses significant antimicrobial activity (De Kimpe et al., 1982); ?-pyrones called umuravumbolide (5,6-dihydro-6-(3-acetoxy-1- heptenyl)-2-pyrone), tetradenolide (5,6-dihydro-6-(1,2-dihydroxyhexyl)-2-pyrone), deacetylmuravumbolide (5,6-dihydro-6-(3-hydroxyl-1-heptenyl)-2-pyrone) and deacetylboronolide (5,6-dihydro-6-(1,2,3-trihydroxyheptyl)-2-pyrone and sitosterol (Zelnik et al., 1978; van Puyvelde et al., 1979; van Puyvelde et al., 1986; van Puyvelde et al., 1987; Davies-Coleman and Rivett, 1995; Scott et al., 2004). 271 A9 Zanthoxylum capense (Thunb.) Harv. Synonyms of Zanthoxylum capense include Fagara capensis Thunb., F. Magaliesmontana Engl., Xanthoxylum (Xanthoxylon) (Waterman, 1975) capense (Thunb.) Harv., Z. thunbergii DC. var obtusifolia Harv. (Hutchings et al., 1996). Common names include Small Knobwood, Adelaide Spice tree, and Cardamon. Other names include Wild cardamom (English); kleinperdepram, Paarde pram, kardamon, knop(pies)doring (Afrikaans); monokwane (Sotho); umlungumabele (Xhosa/Zulu); umnungamabele, amabelentombi, amabelezintshingezi, isimungumabele, isinungwane, umnungwane omncane (Zulu) (Smith, 1895; Hutchings et al., 1996; van Wyk et al., 2009). A9.1 Botanical description of Z. capense Z. capense (Thunb.) Harv. (Rutaceae) is a tree (about 5-10m in height) with many branches. Thick thorns on the bark are a characteristic trait of Z. capense trees. Sharp thorns are also present on the stems. Several pairs of leaflets are found on the leaf (Figure A9.1) with translucent oil glands dotted on the edges. The tree bears fruit, which is said to contain oil, in clusters, which are small and orange brown in colour and resemble oranges (Watt and Breyer-Brandwijk, 1962). The fruit is acrid and tastes of lemon, and presents with a persistent burning sensation in the mouth (Watt and Breyer-Brandwijk, 1962). When ripe, the fruits slit open and reveal shiny black seeds inside. The flowers that are born are greenish-white and are inconspicuous (van Wyk et al., 2009). Root and bark decoctions have a displeasing odour and are said to cause sweating (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). A9.2 Geographical locality of Z. capense Z. capense is distributed widely in the eastern and northern parts of South Africa (van Wyk et al., 2009). Distribution also extends sporadically into the southern regions (Figure A9.2). A9.3 Medicinal uses of Z. capense Z. capense has a reputation of being widely used as a medicine by the African and European people. An infusion of the leaf lends its uses for gastric (Hutchings et al., 1996) and intestinal disorders as well as intestinal parasites (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). The fruit has been used for flatulent colic, palsy, as a stomach remedy and for fever (Watt and Breyer-Brandwijk, 1962; Forbes, 1986; Hutchings et al., 1996; van Wyk et al., 2009). The bark is taken as a tonic in persons suffering from sores (Bryant, 1970; Hutchings 272 et al., 1996) as well as a snakebite treatment. The whole plant is burned on a hot plate and the fumes inhaled for dizziness (Oluwole et al., 2002). Figure A9.1 Leaflets of Z. capense (S.F.van Vuuren). Figure A9.2 Geographical distribution of Z. capense in South Africa (SANBI). A decoction of the bark is given to cattle for gall sickness. It is also used in combination with other medicinal plants to treat tuberculosis, paralysis, epilepsy, infertility and impotency, pleurisy, as an anthelminthic, a purgative, enema, parasiticide as well as for blood purifying (Watt and Breyer-Brandwijk, 1962; Watt, 1967; Hutchings et al., 1996; van Wyk et al., 2009). A decoction of the root is used as a mouthwash (Watt and Breyer-Brandwijk, 1962; Pujol, 1990; Hutchings et al., 1996; Steyn et al., 1998; van Wyk et al., 2009) for aphthae in children and a lotion for acne. Powdered root taken by mouth has been used for pimples and blood poisoning. Powdered bark is used to treat paralysis and relieve toothache by loosening the tooth (Watt and Breyer-Brandwijk, 1962; Hutchings et al., 1996). The root bark as well as a paste of the inner stem bark is similarly used (Smith, 1895; Watt and Breyer-Brandwijk, 1962; Pujol, 1990; Hutchings et al., 1996; van Wyk et al., 2009). Z. capense is widely used to disinfect anthrax-infected meat as well as in the treatment of anthrax (Watt and Breyer- Brandwijk, 1962). 273 Other uses of the root of Z capense include the treatment of bronchitis and for violent chronic coughing when used in combination with other plants (Watt and Breyer-Brandwijk, 1962; Bryant, 1970; Hutchings et al., 1996; Steyn et al., 1998). A9.4 Chemical constituents of Z. capense In 1914, Juritz published the only reported chemical investigation at the time, of Z. capense and it was found that ?a large proportion of a resinous substance was extracted as well as tannins and a yellow colouring substance which was not identified? (Steyn et al., 1998). Since then, alkaloids from African Zanthoxylum capense were isolated (Calderwood et al., 1970; 1971 in Fish and Waterman, 1972). These are skimmianine, chelerythrine, nitidine and N- methyltetrahydropalmatine. In a study by Four compounds isolated from Z. capense were of interest, which were pellitorine, xanthoxylum-?, ?-dimethylallyl ether, ?-sitosterol and sitosterol-?-D-glucose (Steyn et al., 1998). The medicinal properties of Z. capense are said to be supported and explained by the presence of the secondary metabolites pellitorine and sitosterol-?-D-glucose (Steyn et al., 1998). 274 Appendix B ? Conference/publication presentations Publications/conference presentations arising from preliminary studies* Co-author of the following poster: van Vuuren, S.F., Suliman, S., Dhorat, S. and AM Viljoen. The pharmacological interaction of commercial essential oils in combination with conventional antimicrobials. The 38th International Symposium on Essential Oils, Austria, Graz, 9-13 September, 2007. Co-author of the following publication: van Vuuren, S.F., Suliman, S., and Viljoen, A.M., 2009. The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Letters in Applied Microbiology, 48 (4), 440-446. Publications/conference presentations arising from this study* Suliman, S.; van Vuuren, S.F.; Viljoen, A.M. 2008. Validating the antimicrobial efficacy of Artemisia afra in combination with medicinal plants to treat respiratory tract infections. Podium presentation, Academy of Pharmaceutical Sciences of South Africa (PSSA), 2008. Rustenburg, 22-26 September 2008. Young scientist competition presentation. Suliman, S.; van Vuuren, S.F.; Viljoen, A.M. 2009. Antimicrobial interactions of Artemisia afra with other medicinal plants to treat respiratory diseases. Podium presentation, Annual Meeting of the Indigenous Plant Use Forum (IPUF). Stellenbosch, 6-9 July 2009. 3rd Prize winner of young scientist competition presentations. Suliman, S. van Vuuren, S.F.; Viljoen, A.M. 2009. The ethnobotanical study of A. afra in combination therapy for the treatment of respiratory tract infections. Podium presentation at the University of Johannesburg Post Graduated Symposium. Johannesburg, 29 October 2009. Suliman, S., van Vuuren, S.F. and Viljoen, A.M., 2010. Validating the in vitro antimicrobial activity of Artemisia afra in polyherbal combinations to treat respiratory infections. South African Journal of Botany 76, 655-661. *Abstracts can be found in Appendix C 275 Appendix C - Abstracts of presentations/conference presentations 276 Validating the antimicrobial efficacy of Artemisia afra in combination with medicinal plants to treat respiratory tract infections Sajida Suliman 1, Sandy F. van Vuuren 1, Alvaro M. Viljoen 2 1Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193, South Africa. 2Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001, South Africa. Introduction: Artemisia afra is one of the most widely used plants in South African traditional medicine. It is commonly used to treat respiratory infections like coughs, colds, lung inflammation and often combined with plants such as Lippia javanica, Agathosma betulina, Osmitopsis asteriscoides, Eucalyptus globulus, Zanthoxylum capense, Leonotis microphylla, Tetradenia riparia and Allium sativum. The aim of this study is to provide scientific validation on the synergistic, additive and possibly antagonistic interactions between A. afra plant combinations used in traditional medicinal practices. Methods: In vitro minimum inhibitory concentrations (MIC) assays were used to determine antimicrobial efficacy of the essential oil, dichloromethane: methanol (CH2Cl2: MeOH) and aqueous extracts. Calculation of fractional inhibitory concentrations (FIC) and isobologram studies were performed to determine pharmacodynamic interactions, such as synergy, antagonism or additive behaviour. Thin layer chromatography (TLC) combined with bio- autographic assays were used to visualise the antimicrobial activity. Results: MIC?s results were plotted and isobolograms revealed that when Artemisia afra was combined with other plants, mostly additive and synergistic interactions was noted. The essential oil combinations mainly indicated additive interactions. With the aqueous extracts, synergy and antagonism was apparent. The most pronounced synergy was noted for the (CH2Cl2: MeOH) extract of Artemisia afra and Eucalyptus globulus. Synergy was observed 277 against M. catarrhalis, K. pneumoniae, E. faecalis and C. neoformans. TLC and bio- autographic assay results indicate several compounds responsible for antimicrobial activity. Conclusion: Results obtained from antimicrobial assays for various combinations of Artemisia afra with other plants (as prescribed by traditional healers) show varied interactions (synergy, antagonism and additive) depending on the selection of plant combination. 278 Antimicrobial interactions of Artemisia afra with other medicinal plants to treat respiratory diseases. Suliman S.1, S.F. van Vuuren1, A.M. Viljoen2 1Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193. 2Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria 0001. Artemisia afra is one of the most widely used plants in South African traditional medicine. It is commonly used to treat respiratory infections such as coughs, colds, lung inflammation and often combined with plants such as Lippia javanica, Agathosma betulina, Osmitopsis asteriscoides, Eucalyptus globulus, Zanthoxylum capense, Leonotis microphylla, Tetradenia riparia and Allium sativum. The combinations were specifically analysed for any antimicrobial interactions. In addition, the combined use with milk, honey and salt, as traditionally prepared, was evaluated. Methods used included in vitro minimum inhibitory concentrations (MIC) assays, which were used to determine antimicrobial efficacy of the essential oil, dichloromethane: methanol and aqueous extracts. Calculation of fractional inhibitory concentrations (FIC) and isobologram studies were performed to determine pharmacological interactions. The most promising result was the combination of A. afra and A. betulina dichloromethane: methanol extract against M. catarrhalis with a FIC of 0.5. Isobologram results of this combination indicated synergistic, antagonistic and additive pharmacological behaviour against the four pathogens tested, i.e. M. catarrhalis, E. faecalis, K. pneumoniae and C. neoformans. Combinations of A. afra with honey, salt and milk showed mainly indifferent antimicrobial behaviour. An exception was noted i.e. A. afra (aqueous extract) combined with skim milk against M. catarrhalis, indicating additive behaviour with a FIC value of 0.63. Results obtained from the antimicrobial assays for various combinations of A. afra with other plants (as prescribed by traditional healers) show varied interactions depending on the selection of plant combination. 279 The ethnobotanical study of A. afra in combination therapy for the treatment of respiratory tract infections S. (Sajida) Suliman1, S.F. van Vuuren1 & A.M. Viljoen2 1Department of Pharmacy and Pharmacology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Road, Parktown, 2193. 2Department of Pharmaceutical Sciences, Tshwane University of Technology, Private Bag X680, Pretoria, 0001. Artemisia afra is one of the most widely used medicinal plants in South Africa. It is commonly used to treat respiratory infections such as coughs, colds, lung inflammation and often combined with plants such as Lippia javanica, Agathosma betulina, Osmitopsis asteriscoides, Eucalyptus globulus, Zanthoxylum capense, Leonotis microphylla, Tetradenia riparia and Allium sativum. In addition, the combined use with milk, honey and salt is also traditionally prepared. These combinations were evaluated for their antimicrobial interactions. Methods used included in vitro minimum inhibitory concentrations (MIC) assays, which were used to determine antimicrobial efficacy of the essential oil, dichloromethane: methanol and aqueous extracts. Calculation of fractional inhibitory concentration (FIC) and isobolograms demonstrated pharmacological interactions. Results of the combinations of A. afra and L. javanica, and A. afra and O. asteriscoides essential oil, dichloromethane: methanol and aqueous extracts, showed mostly synergistic interactions against all our pathogens tested i.e. M. catarrhalis, E. faecalis, K. pneumoniae and C. neoformans. In the combination of A. afra and A. betulina, and A. afra and Z. capense, varied interactions were noted, with antagonism shown for the aqueous extracts, for all pathogens tested, depending on the ratio in which they were combined. When A. afra and E. globulus was combined mainly synergistic interactions was noted with all pathogens. Combinations of A. afra with honey, salt and milk showed mainly additive and indifferent antimicrobial behaviour. These antimicrobial interactive studies with A. afra demonstrate varied interactions depending on the selection of combination. 280