From Ethnomedicine to Application: Biological Activities and Cytotoxicity of Leaf Extracts from Plants of the Genus Rhododendron by Ahmed Rezk a Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry Approved Dissertation Committee Prof. Dr. Matthias Ullrich, Prof. of Microbiology Prof. Dr. Klaudia Brix, Prof. of Cell Biology Jacobs University Bremen Prof. Dr. Nikolai Kuhnert Prof. of Chemistry Jacobs University Bremen Prof. Dr. Dirk Albach, Prof. of Plant Biodiversity University of Oldenburg Date of Defense: 15.06.2015 This PhD thesis project was financed by Stiftung Rhododendronpark Bremen Dedicated to: My Wife Rasha Acknowledgment Acknowledgment First, I thank Allah for giving me the ability and strength to accomplish this study. I would like to express my gratitude to the following people for support during my work: I would like to express my sincere appreciation and gratitude to my PhD supervisors, Prof. Dr. Matthias Ullrich, and Prof. Dr. Klaudia Brix, who gave me the opportunity to compose my doctoral thesis in their workgroups. I would like to thank them for their support, guidance and all the time they gave to discuss and help in designing experiments to achieve this work. I would also like to thank my dissertation committee members, Prof. Dr. Nikolai Kuhnert and Prof. Dr. Dirk Albach for their time and for their valuable comments during our meetings and reviewing my thesis. I would specifically like to thank AG Ullrich and AG Brix lab members, Amna Mehmood, Antje Stahl, Gabriela Alfaro-Espinoza, Khaled Abdallah, Neha Kumari, Maria Qatato, Joanna Szumska, and Jonas Weber for maintaining a friendly and family working environment. I want to thank my lab rotation students Alaa Al-Hashimi and Warren John for their contributions in my project. Sincere thanks for Maren Rehders, who guided me in all cell biology methods and her great help for discussing results and all technical issues in the lab. I cannot forget our great team assistants Maike Last and Nina Böttcher for their technical helps. I am thankful to the Stiftung Bremer Rhododendronpark and Jacobs University Bremen for providing the financial support throughout the period of my studies. I am particularly grateful to Mr. Wolfgang Klunker for his enthusiastic support. I cannot forget to thank Dr. Hartwig Schepker for providing plant materials and discuss the original habitat of Rhododendron. Last but not least, I have great pleasure to express my deep sense of gratitude towards the woman who supported and stood by me in all circumstances, my wife Rasha El-Abassy. A very warm and affectionate thanks to my lovely daughters Judy and Lena. 3 | P a g e Table of contents Table of Contents Acknowledgment ................................................................................................................................... 3 Abstract .................................................................................................................................................. 6 1. Introduction ................................................................................................................................... 8 1.1. Historical Background ............................................................................................................. 8 1.2. Rhododendron Classification and Description ........................................................................ 8 1.3. Habitat and distribution of genus Rhododendron .................................................................. 10 1.4. Use of Rhododendron in Traditional Medicine ..................................................................... 11 1.5. Plant metabolites ................................................................................................................... 14 1.6. Plant Secondary Metabolites as Drugs .................................................................................. 16 1.7. Antibiotics and Bacterial Defense Mechanisms .................................................................... 19 1.8. Drug development ................................................................................................................. 21 1.9. Cell death ............................................................................................................................... 22 1.9.1 Necrosis ......................................................................................................................... 23 1.9.2 Apoptosis ....................................................................................................................... 23 2. Aims of the study ......................................................................................................................... 28 3. Results........................................................................................................................................... 29 3.1 ....... Phylogenetic spectrum and analysis of antibacterial activities of leaf extracts from plants of the genus Rhododendron ............................................................................................................................. 31 3.2 ........ Assessment of Cytotoxicity Exerted by Leaf Extracts from Plants of the Genus Rhododendron towards Epidermal Keratinocytes and Intestine Epithelial Cells .......................................................... 56 3.3 .............Distinguishing the polyphenolic and antibacterial profile of the leaves, fruits and flowers of Rhododendron ambiguum and Rhododendron cinnabarinum using high performance liquid chromatography coupled with ion trap and time of flight mass spectrometry .................................... 102 4. Discussion ................................................................................................................................... 119 5. References .................................................................................................................................. 125 6. Appendix .................................................................................................................................... 139 5 | P a g e Abstract Abstract The evolution of bacterial resistance to current antibiotics is one of the biggest threats to human health. There is an increasing interest to identify novel antimicrobial compounds from various natural sources. Plants-derived compounds are used as ingredients in traditional treatment of numerous human disorders including infectious diseases caused by pathogenic microorganisms. In planta, secondary metabolites like the polyphenols are known to act as potent antimicrobial agents against several plant pathogens. Plants of the genus Rhododendron are typically used in a range of ethno-medical applications. There are more than 1,000 Rhododendron species growing/spreading/distributed particularly in the Northern hemisphere. The City of Bremen harbors the Rhododendron-Park in which approximately 600 different species of Rhododendron and hybrids are grown. This enables research with about two thirds of all known Rhododendron species. The aim of this study is to identify novel compounds from the secondary metabolite pool synthesized by Rhododendron that can be used as antimicrobial treatments of human diseases/maladies in form of ectopic application or as orally administered drugs. Therefore, leaf extracts of a total of 120 different Rhododendron species were tested using the agar diffusion assay towards twenty-six bacterial species representing different taxonomic clades of non- pathogenic strains Gram-positive and Gram-negative bacteria. The possible cytotoxic effects of the most promising, antimicrobial bioactive extracts from Rhododendron species were assessed in a concentration-dependent manner using epidermal keratinocytes of the skin and epithelial cells of the intestinal mucosa, respectively, as target cell systems. The leaf extracts of 17 Rhododendron species exhibited significant growth-inhibiting activities against Gram-positive bacteria. In contrast, only very few of the leaf extracts affected the growth of Gram-negative bacteria. All leaf extracts with antimicrobial bioactivity were extracted from representatives of the subgenus Rhododendron, with 15 from the sub-section Rhododendron and two belonging to the section Pogonanthum. Six Rhododendron species out of the 17 species showed the lowest minimum inhibitory concentration with 50 µg leaf extract powder per mL solvent. Equally low and moderate concentrations (50 µg/ml) of leaf extracts from three of these plant species were non-toxic towards both mammalian cell lines, i.e. HaCaT keratinocytes and IEC6 enterocytes. However, higher doses such as 500 µg/mL of Rhododendron leaf extracts were potent in negatively affecting both, keratinocytes and, particularly, the intestine epithelial cells. 6 | P a g e Abstract We conclude that bioactive compounds with high antimicrobial activities can be extracted from the leaves of Rhododendron species mainly belonging to the subgenus Rhododendron, and that they acted mostly against Gram-positive organisms. The leaf extracts from R. minus, R. ferrugineum, and R. racemosum applied at a concentration of 50 µg/ml proved safe to be used in 24-h-incubations of monolayer cultures of both, HaCaT keratinocytes and IEC6 intestine epithelial cells. In contrast, high doses of most leaf extracts induced apoptosis evidenced by a significant increase in the levels of active caspase-3 in IEC6 intestine epithelial cells.