University of Szeged Faculty of Pharmacy Graduate School of Pharmaceutical Sciences Department of Pharmacognosy Biologically active secondary metabolites from Asteraceae and Polygonaceae species Ph.D. Thesis Ildikó Lajter Supervisors: Prof. Judit Hohmann Dr. Andrea Vasas Szeged, Hungary 2015 LIST OF PUBLICATIONS RELATED TO THE THESIS I. Lajter I, Zupkó I, Molnár J, Jakab G, Balogh L, Vasas A, Hohmann J. Antiproliferative activity of Polygonaceae species from the Carpathian Basin against human cancer cell lines Phytotherapy Research 2013; 27: 77-85. II. Lajter I, Vasas A, Orvos P, Bánsághi S, Tálosi L, Jakab G, Béni Z, Háda V, Forgo P, Hohmann J. Inhibition of G protein-activated inwardly rectifying K+ channels by extracts of Polygonum persicaria and isolation of new flavonoids from the chloroform extract of the herb Planta Medica 2013; 79: 1736-1741. III. Lajter I, Vasas A, Béni Z, Forgo P, Binder M, Bochkov V, Zupkó I, Krupitza G, Frisch R, Kopp B, Hohmann J. Sesquiterpenes from Neurolaena lobata and their antiproliferative and anti-inflammatory activities Journal of Natural Products 2014; 77: 576-582. IV. McKinnon R, Binder M, Zupkó I, Afonyushkin T, Lajter I, Vasas A, de Martin R, Unger C, Dolznig H, Diaz R, Frisch R, Passreiter CM, Krupitza G, Hohmann J, Kopp B, Bochkov VN. Pharmacological insight into the anti-inflammatory activity of sesquiterpene lactones from Neurolaena lobata (L.) R.Br. ex Cass. Phytomedicine 2014; 21: 1695-1701. V. Lajter I, Pan SP, Nikles S, Ortmann S, Vasas A, Csupor-Löffler B, Forgó P, Hohmann J, Bauer R. Inhibition of COX-2 and NF-κB1 gene expression, NO production, 5-LOX, and COX-1 and COX-2 enzymes by extracts and constituents of Onopordum acanthium Planta Medica 2015; 81: 1270-1276. TABLE OF CONTENTS ABBREVIATIONS AND SYMBOLS ...................................................................................................................... 1 1. INTRODUCTION .................................................................................................................................... 2 2. AIMS OF THE STUDY .............................................................................................................................. 4 3. LITERATURE OVERVIEW .......................................................................................................................... 5 3.1. Botany ....................................................................................................................................... 5 3.1.1. Botany of the family Asteraceae and the investigated species ........................................... 5 3.1.2. Botany of the family Polygonaceae and the investigated species ...................................... 5 3.2. Chemistry .................................................................................................................................. 6 3.2.1. Chemistry of the family Asteraceae and the investigated species ...................................... 6 3.2.2. Chemistry of the family Polygonaceae and the investigated species ................................. 7 3.3. Folk-medicinal uses of the investigated plant species ............................................................. 8 3.4. Pharmacology ........................................................................................................................... 9 3.4.1. Pharmacology of the family Asteraceae and the investigated species ............................... 9 3.4.2. Pharmacology of the family Polygonaceae and the investigated species ......................... 12 4. MATERIALS AND METHODS .................................................................................................................. 14 4.1. Plant material .......................................................................................................................... 14 4.2. Extraction ................................................................................................................................ 15 4.2.1. Preparation of extracts for pharmacological screening .................................................... 15 4.2.2. Extraction of the plant materials for preparative phytochemical work ............................ 15 4.3. Purification and isolation of compounds ................................................................................ 15 4.3.1. Open-column chromatography (OCC) ............................................................................... 15 4.3.2. Vacuum-liquid chromatography (VLC)............................................................................... 16 4.3.3. Rotation planar chromatography (RPC)............................................................................. 16 4.3.4. Medium-pressure liquid chromatography (MPLC) ............................................................ 17 4.3.5. Preparative layer chromatography (PLC) .......................................................................... 17 4.3.6. Gel filtration (GF) ............................................................................................................... 17 4.3.7. High-performance liquid chromatography (HPLC) ............................................................ 18 4.4. Structure determination of the isolated compounds ............................................................. 18 4.5. LC-MS investigation of P. persicaria samples ......................................................................... 18 4.6. Pharmacological tests ............................................................................................................. 19 4.6.1. In vitro antiproliferative investigations ............................................................................. 19 4.6.2. GIRK channel-inhibitory assay ........................................................................................... 19 4.6.3. In vitro and in vivo anti-inflammatory investigations ........................................................ 20 5. RESULTS ............................................................................................................................................ 22 5.1. Screening of Polygonaceae species for antiproliferative and GIRK channel inhibitory activities .................................................................................................................................. 22 5.2. Investigation of N. lobata, O. acanthium and P. persicaria extracts for bioactivity ............... 23 5.3. Isolation of compounds from Neurolaena lobata .................................................................. 24 5.4. Isolation of compounds from Onopordum acanthium ........................................................... 26 5.5. Isolation of compounds from Polygonum persicaria .............................................................. 27 5.6. Characterization and structure determination of the isolated compounds .......................... 28 6. DISCUSSION ....................................................................................................................................... 37 6.1. Screening studies .................................................................................................................... 37 6.2. Investigation of N. lobata, O. acanthium and P. persicaria .................................................... 38 6.2.1. Isolation of bioactive compounds...................................................................................... 38 6.2.2. Structure elucidation ......................................................................................................... 40 6.2.3. Biological activities ............................................................................................................ 41 7. SUMMARY ......................................................................................................................................... 47 8. REFERENCES ....................................................................................................................................... 49 ACKNOWLEDGEMENTS ABBREVIATIONS AND SYMBOLS 1D one-dimensional 2D two-dimensional APCI atmospheric pressure chemical ionization COSY correlated spectroscopy EIMS electron ionization mass spectrometry ESIMS electron spray ionization mass spectrometry GF gel filtration GIRK G protein-activated inwardly rectifying K+ channel HMBC heteronuclear multiple-bond correlation spectroscopy HPLC high-performance liquid chromatography HRE(S)IMS high-resolution electron (spray) ionization mass spectrometry HRMS high-resolution mass spectrometry HSQC heteronuclear single-quantum coherence spectroscopy JMOD J-modulated spin-echo experiment MPLC medium-pressure liquid chromatography MS mass spectrometry NF-ĸB nuclear factor-ĸB NMR nuclear magnetic resonance NO nitric oxide NOE nuclear Overhauser effect NOESY nuclear Overhauser enhancement spectroscopy NP normal-phase NSAID non-steroidal anti-inflammatory drug OCC open-column chromatography PLC preparative-layer chromatography RP reversed-phase RPC rotation planar chromatography SL sesquiterpene lactone TLC thin-layer chromatography TMS tetramethylsilane UV ultraviolet VLC vacuum-liquid chromatography δ chemical shift 1 1. INTRODUCTION Cancer and cardiovascular diseases are the leading causes of death in the western world.1 Cancer and certain cardiovascular diseases, such as atherosclerosis, are often associated with inflammation, and it has been demonstrated that chronic inflammation may be the common factor in many diseases.2 The clinically