Recovery of new diarylheptanoid sources in Betulaceae Characterisation of the phenolic profile of Corylus species by HPLC-ESI-MS methods Ph.D. Dissertation Eszter Riethmüller Semmelweis University Doctoral School of Pharmaceutical Sciences Supervisor: Dr. Ágnes Kéry, Ph.D. Reviewers: Dr. Gergely Völgyi Ph.D. Dr. Attila Hunyadi Ph.D. Chair of final examination committee: Dr. Éva Lemberkovics Ph.D. Members of final examination committee: Dr. Huba Kalász D.Sc. Dr. Imre Máthé D.Sc. Budapest 2016 TABLE OF CONTENTS TABLE OF CONTENTS ......................................................................................................... 2 1. LIST OF ABBREVIATIONS ............................................................................................... 6 2. INTRODUCTION ............................................................................................................. 8 2.1. Structural features and biosynthesis of diarylheptanoids ............................. 10 2.2. Distribution of diarylheptanoids in the plant kingdom................................. 12 2.3. Biological activities of diarylheptanoids ...................................................... 14 2.3.1. Curcuma genus (Zingiberaceae)................................................................... 14 2.3.1.1. Structural features and bioavailability of curcumin ..................................... 16 2.3.1.2. Antioxidant activity of curcumin.................................................................. 16 2.3.1.3. Anti-inflammatory effect of curcumin ......................................................... 17 2.3.1.4. Anti-tumoural properties of curcumin .......................................................... 19 2.3.1.5. The positive effects of curcumin in neurodegenerative diseases ................. 20 2.3.1.6. The dark side of curcumin ............................................................................ 21 2.3.2. Alpinia genus (Zingiberaceae)...................................................................... 22 2.3.2.1. Effect on neuronal differentiation ................................................................ 23 2.3.2.2. Selective cytotoxic effect ............................................................................. 23 2.3.2.3. Antibacterial effect ....................................................................................... 24 2.3.2.4. Platelet-activating factor receptor binding inhibitory activity...................... 24 2.3.2.5. Anti-angiogenic activity ............................................................................... 24 2.3.2.6. Anti-inflammatory activity ........................................................................... 25 2.3.3. Morella and Myrica genera (Myricaceae) .................................................... 25 2.3.3.1. Antioxidant activity ...................................................................................... 25 2.3.3.2. Anti-inflammatory activity ........................................................................... 26 2.3.3.3. Anticancer activity........................................................................................ 26 2.3.3.4. Positive effect in neurodegenerative diseases .............................................. 27 2.3.4. Acer genus (Aceraceae) ................................................................................ 27 2.3.4.1. Anti-inflammatory effect .............................................................................. 27 2.3.4.2. Effect on osteoblast differentiation .............................................................. 27 2 2.3.4.3. Anti-diabetic effect ....................................................................................... 28 2.3.5. Juglans genus (Juglandaceae) ...................................................................... 28 2.3.5.1. Neuroprotective and antioxidant effect ........................................................ 28 2.3.6. Alnus genus (Betulaceae) ............................................................................. 29 2.3.6.1. Antioxidant activity ...................................................................................... 29 2.3.6.2. Anticancer activity........................................................................................ 30 2.3.6.3. Anti-inflammatory activity ........................................................................... 31 2.3.6.4. Immunosuppressive activity ......................................................................... 31 2.3.6.5. Other effects ................................................................................................. 32 2.3.7. Betula genus (Betulaceae) ............................................................................ 32 2.3.7.1. Antifibrotic activity ...................................................................................... 32 2.3.7.2. Selective cytotoxic activity........................................................................... 33 2.4. Corylus avellana L. (Common hazel, Betulaceae)....................................... 33 2.4.1. Ethimology ................................................................................................... 33 2.4.2. Taxonomic classification .............................................................................. 33 2.4.3. Morphology .................................................................................................. 34 2.4.4. Traditional use .............................................................................................. 34 2.4.5. Pharmacological effects................................................................................ 35 2.4.6. Phytochemical characterisation .................................................................... 36 2.5. Corylus colurna L. (Turkish hazel, Betulaceae) .......................................... 37 2.5.1. Morphology .................................................................................................. 37 2.5.2. Traditional use .............................................................................................. 38 2.5.3. Pharmacological effects................................................................................ 38 2.5.4. Phytochemical characterisation .................................................................... 39 2.6. Corylus maxima Mill. (Filbert, Betulaceae) ................................................. 39 2.6.1. Ethymology .................................................................................................. 39 2.6.2. Morphology .................................................................................................. 39 2.6.3. Traditional use .............................................................................................. 40 2.6.4. Phytochemical characterisation .................................................................... 40 3. OBJECTIVES ................................................................................................................ 41 4. MATERIALS AND METHODS ......................................................................................... 43 4.1. Plant material ................................................................................................ 43 3 4.2. Solvents and chemicals................................................................................. 43 4.3. Extraction and sample preparation ............................................................... 44 4.4. Quantitative phytochemical analyses ........................................................... 44 4.5. Antioxidant activity assays ........................................................................... 44 4.6. Characterisation of phenolics in the Corylus extracts by HPLC-DAD-MS . 45 4.6.1. HPLC-DAD-QMS conditions ...................................................................... 45 4.6.2. HPLC-DAD-TOF-MS conditions ................................................................ 46 4.6.3. HPLC-DAD-MS/MS conditions .................................................................. 48 4.7. Quantitative analyses by HPLC-MS/MS...................................................... 49 4.7.1. Determination of diarylheptanoids ............................................................... 49 4.7.2. Determination of flavonoids ......................................................................... 49 4.7.3. Method validation – Calibration, precision, accuracy and quality control .. 50 4.8. HPLC-based DPPH scavenging assay.......................................................... 51 4.8.1. Sample preparation ....................................................................................... 51 5. RESULTS ..................................................................................................................... 52 5.1. Quantitative phytochemical analyses ........................................................... 52 5.2. Antioxidant activity assays ........................................................................... 52 5.2.1. Scavenging activity on DPPH free radical ................................................... 53 5.2.2. Scavenging activity on ABTS free radical ................................................... 54 5.3. Characterisation of phenolics in the Corylus extracts by HPLC-MS ........... 55 5.3.1. Corylus avellana L. ...................................................................................... 56 5.3.2. Corylus colurna L........................................................................................