CZECH UNIVERSITY OF LIFE SCIENCES PRAGUE Faculty of Tropical AgriSciences Department of Crop Sciences and Agroforestry Antimicrobial effect of medicinal plants used in traditional Indonesian medicine DOCTORAL THESIS Author: Andreas Romulo Supervisor: prof. Ing. Ladislav Kokoška, Ph.D. 2018 ACKNOWLEDGMENT I would like to express my gratitude towards my exceptional supervisor prof. Ladislav Kokoška, Ph.D. for his sincere guidance, encouragement, and patience over the last three years. Thank you for giving me the opportunity to do research in the Laboratory of Ethnobotany and Ethnopharmacology. Your continuous support, insightful comments, suggestion, and critical analysis of the research and writing of this dissertation were essential for completion of my study and the success of my future career. I would like to thank Ing. Johana Rondevaldová, Ph.D. for the support and helpful assistance in laboratory research and academic background, as well as to my fellow labmates, Ing. Markéta Houdková and Ing. Marie Netopilová who helped and contributed in the experiment. My special thanks to Ing. Klára Urbanová, Ph.D. and Ing. Pavel Nový, Ph.D. for their sincere help, guidance, and suggestion during the analysis of the essential oil. Finally, I would like to thank to my beloved parents and friends for their continues support, encouragement, and pray. I am so grateful to have you all. This research was financially supported by Erasmus Mundus project ALFABET (Asia: Life, Food, Agriculture, Biology, Economics, Technology) [55207], Czech University of Life Sciences Prague Grant Agency project CIGA [20175001] and Internal Grant Agency project IGA [20175020]. II CERTIFICATION I, Andreas Romulo, submitted this dissertation for Ph.D. degree at the Czech University of Life Sciences Prague, Faculty of Tropical AgriSciences, declare that this dissertation is my own work unless otherwise referenced or acknowledged. In Prague, 10 August 2018 .............................................. Andreas Romulo, M.Sc. III TABLE OF CONTENTS ACKNOWLEDGMENT ............................................................................................................... II CERTIFICATION ....................................................................................................................... III TABLE OF CONTENTS ............................................................................................................ IV LIST OF TABLES ........................................................................................................................V LIST OF FIGURES .......................................................................................................................V LIST OF APPENDICES ............................................................................................................. VI LIST OF ABBREVIATION ...................................................................................................... VII ABSTRACT ..............................................................................................................................VIII ABSTRAKT ................................................................................................................................ IX ABSTRAK ....................................................................................................................................X 1. INTRODUCTION ................................................................................................................. 1 2. REVIEW OF LITERATURE ................................................................................................ 3 2.1. Microbial infections ...................................................................................................... 3 2.2. Microorganisms ............................................................................................................. 4 2.2.1. Candida albicans ....................................................................................................... 5 2.2.2. Enterococcus faecalis ................................................................................................ 6 2.2.3. Escherichia coli ......................................................................................................... 8 2.2.4. Pseudomonas aeruginosa .......................................................................................... 9 2.2.5. Staphylococcus aureus ............................................................................................ 10 2.3. Ethnopharmacological approach to anti-infective drugs discovery ............................ 13 2.4. Traditional Indonesian medicine (Jamu) ..................................................................... 17 2.4.1. Characteristics of selected studied plant species ..................................................... 20 3. HYPOTHESIS ..................................................................................................................... 27 4. OBJECTIVES ...................................................................................................................... 28 5. MATERIAL AND METHODS ........................................................................................... 29 5.1. Plant materials ............................................................................................................. 29 5.2. Preparation of plant extract ......................................................................................... 29 5.3. Essential oil isolation .................................................................................................. 29 5.4. Microorganisms and media ......................................................................................... 29 5.5. Broth microdilution assay ........................................................................................... 34 5.6. Modified broth microdilution assay ............................................................................ 35 5.7. Broth microdilution volatilisation assay ...................................................................... 35 5.8. GC and GC-MS analysis ............................................................................................. 36 6. RESULTS AND DISCUSSION .......................................................................................... 37 6.1. Screening of in vitro antimicrobial activity of plant extracts ...................................... 37 6.2. Assessment of antimicrobial and antistaphylococcal effect of C. mangga EO ........... 42 6.3. Analysis of chemical composition of C. mangga EO ................................................. 44 7. CONCLUSION .................................................................................................................... 53 8. REFERENCES .................................................................................................................... 54 9. APPENDICES ..................................................................................................................... 81 IV LIST OF TABLES Table 1. Ethnobotanical data on Indonesian medicinal plants tested .............................. 31 Table 2. Antimicrobial activity of ethanol extracts from Indonesian medicinal plants .. 40 Table 3. In vitro antimicrobial activity of Curcuma mangga rhizome essential oil in liquid and vapour phase ............................................................................................... 43 Table 4. In vitro antistaphylococcal activity of Curcuma mangga rhizome essential oil ....................................................................................................................... 43 Table 5. Chemical composition of Curcuma mangga rhizome essential oil ................... 45 LIST OF FIGURES Figure 1. Aleurites moluccanus. ..................................................................................... 81 Figure 2. Curcuma mangga ............................................................................................ 81 Figure 3. Orthosiphon aristatus ..................................................................................... 81 Figure 4. Rotheca serrata ............................................................................................... 82 Figure 5. Woodfordia floribunda .................................................................................... 82 Figure 6. FID chromatogram C. mangga rhizome EO on HP-5MS column. ................. 83 Figure 7. FID chromatogram C. mangga rhizome EO on DB-17MS column. .............. 84 Figure 8. Total ion current (TIC) chromatogram of C. mangga rhizome EO on HP-5MS column obtained from GC-QTOF-MS ........................................................... 85 Figure 9. Mass spectrum of (E)-15,16-dinorlabda-8(17),11-dien-13-one ...................... 86 Figure 10. Mass spectrum of ambrial ............................................................................. 86 Figure 11. Mass spectrum of 13-nor-eremophil-1(10)-en-11-one.................................. 87 Figure 12. Mass sprectrum of (E)-15,16-dinorlabda-8(17),12-dien-14-al ..................... 87 Figure 13. Mass sprectrum of aromadendrene oxide ..................................................... 88 V LIST OF APPENDICES Appendix A. Photographic illustrations of plant used in traditional Indonesian medicine .................................................................................................... 81 Appendix B. Chromatograms of C. mangga rhizome EO .............................................