Finding bioactive compounds in plant extracts by HPLC-coupled assays: novel approaches to natural product-based drug discovery Ph.D. Thesis Árpád Könczöl Semmelweis University Doctoral School of Pharmaceutical Sciences Supervisor: Dr. György Tibor Balogh, Ph.D. Consultant: Dr. Ágnes Kéry, Ph.D. Official Reviewers: Dr. Huba Kalász, D.Sc. Dr. Dezső Csupor, Ph.D. Chair of Exam Committee: Dr. Kornélia Tekes, Ph.D. Exam Committee: Dr. Éva Lemberkovics, Ph.D. Dr. József Balla, Ph.D. Budapest, 2013 Contents CONTENTS .................................................................................................................... 2 ABBREVIATIONS AND SYMBOLS .......................................................................... 5 1. INTRODUCTION ...................................................................................................... 7 1.1. Paradigm and challenges of the current drug discovery ........................................ 7 1.2. Status and relevance of natural products in drug discovery .................................. 8 1.3. Novel approaches to natural product lead finding: profiling by coupled techniques ................................................................................................................... 11 1.4. Review of targets and methods used in the screening and profiling of the plant extract library .............................................................................................................. 15 1.4.1. Free radicals, oxidative stress and antioxidants ........................................... 16 1.4.1.1. The DPPH (2,2-diphenyl-1-picrylhydrazyl) method ............................. 18 1.4.1.2. The peroxynitrite anion (ONOO–): biochemistry and methodologies for measurement of ONOO– scavenging activity ..................................................... 21 1.4.2. The blood-brain barrier (BBB) ..................................................................... 23 1.4.2.1. Methodologies for measurement of blood-brain barrier transport ........ 25 1.4.2.2. The parallel artificial membrane permeability assay for blood-brain barrier (PAMPA-BBB) ....................................................................................... 26 1.4.2.3. Rationale of studying natural products and plant extracts by PAMPA- BBB .................................................................................................................... 27 2. OBJECTIVES ........................................................................................................... 28 3. MATERIALS AND METHODS ............................................................................. 30 3.1. Chemicals and reference compounds .................................................................. 30 3.2. Plant material ....................................................................................................... 30 3.2.1. Plant extract library ...................................................................................... 30 3.2.2. Artemisia gmelinii Webb. ex Stechm. (Asteraceae) ..................................... 31 3.2.3. Salvia miltiorrhiza Bunge (Lamiaceae) ........................................................ 32 3.2.4. Tanacetum parthenium (L.) Sch. Bip. (Asteraceae) ..................................... 32 3.2.5. Corydalis cava (L.) Schweig. & Kört. (Papaveraceae) ................................ 33 3.2.6. Salvia officinalis L. (Lamiaceae) .................................................................. 33 3.2.7. Vinca major L. (Apocynaceae) ..................................................................... 34 3.3. Instrumentation .................................................................................................... 34 3.3.1. High performance liquid chromatography – mass spectrometry ................. 34 3.3.2. Nuclear magnetic resonance spectroscopy ................................................... 35 3.4. Cytotoxicity screening campaign ........................................................................ 35 3.4.1. Cell line ........................................................................................................ 35 2 3.4.2. Procedure of screening ................................................................................. 36 3.4.3. Data handling ................................................................................................ 36 3.5. Antioxidant activity screening campaign ............................................................ 37 3.5.1. Procedure of screening ................................................................................. 37 3.5.2. Data handling and IC50 measurement ........................................................... 38 3.6. Antioxidant activity-guided phytochemical investigation of Artemisia gmelinii 39 3.6.1. HPLC-based DPPH scavenging assay .......................................................... 39 3.6.2. Isolation of compounds 7a and 8a with preparative HPLC ......................... 39 3.6.3. High resolution mass spectrometry analysis ................................................ 40 3.6.4. NMR spectroscopy ....................................................................................... 40 3.7. HPLC-based peroxynitrite scavenging assay ...................................................... 41 3.7.1. Synthesis of the peroxynitrite anion ............................................................. 41 3.7.2. Preparation of the Salvia specific model mixture ......................................... 41 3.7.3. HPLC conditions .......................................................................................... 41 3.7.4. Peroxynitrite scavenging activity measurement in 96-well plate ................. 42 3.8. Blood-brain barrier permeability screening campaign and related methods ....... 43 3.8.1. PAMPA-BBB procedures ............................................................................ 43 3.8.2. HPLC-MS analysis ....................................................................................... 44 3.8.3. Gas chromatography – flame ionization detector analysis of PAMPA-BBB co-solvents .............................................................................................................. 46 3.8.4. NMR spectroscopy ....................................................................................... 46 3.9. Statistical analysis................................................................................................ 47 4. RESULTS .................................................................................................................. 48 4.1. Cytotoxicity screening ......................................................................................... 48 4.2. Antioxidant activity screening ............................................................................. 49 4.3. HPLC-based antioxidant activity profiling of the methanolic extract of Artemisia gmelinii ....................................................................................................................... 51 4.3.1. Introduction .................................................................................................. 51 4.3.2. Phytochemical and antioxidant characterization .......................................... 52 4.4. HPLC-based peroxynitrite scavenging activity profiling of Salvia spp. ............. 56 4.4.1. Introduction .................................................................................................. 56 4.4.2. Optimization of the chromatographic conditions ......................................... 59 4.4.3. Calibration for pyrogallol red ....................................................................... 60 4.4.4. Validation of the assay with a Salvia specific model mixture ...................... 61 4.4.4.1. Comparison of degradation kinetics in mixture to individual scavenging activities .............................................................................................................. 63 4.4.4.2. Validation of the assay parameters with structure – activity relationships ............................................................................................................................ 65 4.4.5. Demonstration of the assay performance on the methanolic extract of Salvia miltiorrhiza ............................................................................................................. 65 3 4.5. Blood-brain barrier (BBB) permeability screening and HPLC-based hit profiling .................................................................................................................................... 67 4.5.1. Introduction .................................................................................................. 67 4.5.2. Validation of the PAMPA-BBB assay for natural products ......................... 68 4.5.3. Characterization of the effective BBB-permeability potential of major phytochemical compound classes ........................................................................... 69 4.5.4. Co-solvent retention profile of the PAMPA-BBB assay .............................. 71 4.5.5. Screening of the plant extract library and tentative physicochemical characterization of BBB+ and BBB- plant extracts by LC-MS ............................. 72 4.5.6. Application of the PAMPA-BBB/LC-MS/NMR procedure to four BBB+ plant extracts ........................................................................................................... 74 4.5.7. Evaluating the CNS-activity of the identified