HISTOLOGICAL, PHYTOCHEMICAL AND ANTIMICROBIAL EVALUATION OF COFFEA SPECIES Ph.D. dissertation Éva Brigitta Patay Faculty of Pharmacy University of Pécs Pécs 2017 Histological, phytochemical and antimicrobial evaluation of Coffea species Ph.D. dissertation Éva Brigitta Patay Supervisor: dr. Nóra Papp PhD Program leader: dr. József Deli doctor of HAS, full professor Head of the Doctoral School: dr. Erika Pintér full professor Department of Pharmacognosy Faculty of Pharmacy University of Pécs, Hungary Pécs, 2017 Table of Contents 1. Introduction ............................................................................................................................... 5 2. Literature review ........................................................................................................................ 7 2.1. Description and synonyms of coffee names .......................................................................... 7 2.2. Historical description and discovery of Coffea species ......................................................... 7 2.3. Taxonomy .......................................................................................................................... 10 2.4. Geographical distribution ................................................................................................... 13 2.5. Morphological description.................................................................................................. 15 2.6. Histological description ...................................................................................................... 17 2.7. Molecular biological characters .......................................................................................... 17 2.8. Reproduction, cultivation, processing ................................................................................. 19 2.9. Pests................................................................................................................................... 21 2.10. Phytochemical analyses of plant parts of coffees .............................................................. 24 2.10.1. Coffee seed ............................................................................................................... 24 2.10.2. Coffee leaf ................................................................................................................ 26 2.10.3. Coffee coal................................................................................................................ 28 2.11. Histological localisation of metabolites ............................................................................ 29 2.12. Adaptation to inadequate environmental conditions .......................................................... 30 2.13. Ethnobotanical data .......................................................................................................... 30 2.14. Medicinal importance and application of coffees .............................................................. 31 2.14.1. Adverse effects of coffee........................................................................................... 35 2.14.2. Interactions ............................................................................................................... 36 2.14.3. Caffeine-free and decaffeinated products .................................................................. 37 3. Histological study of the selected Coffea species ...................................................................... 38 3.1. Materials and methods ....................................................................................................... 38 3.1.1. Chemicals ................................................................................................................... 38 3.1.2. Sample preparation ..................................................................................................... 38 3.1.3. Leaf clearing method .................................................................................................. 40 3.1.4. Embedding and preparation of the studied plant parts .................................................. 40 3.1.5. Statistical analysis ....................................................................................................... 41 3.2. Results and discussion ........................................................................................................ 41 2 3.2.1. Characters of cleared leaves ........................................................................................ 41 3.2.2. Histological features on preparations ........................................................................... 42 3.2.2.1. Histology of the leaves ........................................................................................ 42 3.2.2.2. Histology of the petioles ...................................................................................... 46 3.2.2.3. Histology of the stems ......................................................................................... 48 3.2.2.4. Histology of the fruits .......................................................................................... 50 3.2.3 Conclusions ................................................................................................................. 54 4. Phytochemical study of the selected Coffea species .................................................................. 55 4.1. Materials and methods ....................................................................................................... 56 4.1.1. Chemicals ................................................................................................................... 56 4.1.2. Sample preparation ..................................................................................................... 56 4.1.3. HPLC-ESI/MS ............................................................................................................ 57 4.1.4. HPLC-ESI-MS/MS ..................................................................................................... 58 4.1.5. Total polyphenol and tannin content............................................................................ 59 4.2. Results and discussion ........................................................................................................ 60 4.2.1. Compounds detected by HPLC-ESI/MS ...................................................................... 60 4.2.2. Compounds detected by HPLC-ESI-MS/MS ............................................................... 62 4.2.3. Total polyphenol and tannin content............................................................................ 67 4.2.4. Conclusions ................................................................................................................ 69 5. Antioxidant activity of the selected Coffea species ................................................................... 70 5.1. Materials and methods ....................................................................................................... 72 5.1.1. Chemicals ................................................................................................................... 72 5.1.2. Sample preparation ..................................................................................................... 72 5.1.3. 2,2-diphenyl-1-picrylhydrazyl method (DPPH) ........................................................... 72 5.1.4. Enhanced chemiluminescence method (ECL).............................................................. 73 5.1.5. Oxygen radical absorbance capacity assays (ORAC) ................................................... 73 5.1.6. Instrumentation and interpretation of data of antioxidant tests (ECL, DPPH, ORAC) .. 74 5.2. Results and discussion ........................................................................................................ 75 5.2.1. Results of the applied antioxidant tests ........................................................................ 75 5.2.2. Conclusions ................................................................................................................ 77 6. Antimicrobial activity of the selected Coffea species ................................................................ 78 3 6.1. Materials and methods ....................................................................................................... 80 6.1.1. Chemicals ................................................................................................................... 80 6.1.2.Sample preparation ...................................................................................................... 80 6.1.3. Disc diffusion and agar diffusion methods .................................................................. 80 6.2. Results and discussion ........................................................................................................ 81 6.2.1. Disc diffusion method ................................................................................................. 81 6.2.2. Agar diffusion method ................................................................................................ 82 6.2.3 Conclusions............................................................................................................. ........ 83 7. Summary and novel findings .................................................................................................... 84 8. References ..............................................................................................................................