PHARMACEUTICAL VALUE OF ONIONS (ALLIUM L.) AND RELATED SPECIES OF CENTRAL ASIA Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Dem Fachbereich Pharmazie der Philipps-Universität Marburg vorgelegt von Jarmila Jedelská aus Šternberk Marburg/Lahn 2007 Vom Fachbereich Pharmazie der Philipps-Universität Marburg als Dissertation angenommen am ………………………. Erstgutachter: Doc. RNDr. Petr Solich CSc. Zweitgutachter: Prof. Dr. Udo Bakowsky Tag der mündlichen Prüfung am 18.04.2007 Mým rodičům a blízkým List of Contents List of Contents List of Contents ........................................................................................................................1 Acknowledgements ..................................................................................................................3 List of Abbreviations .................................................................................................................5 1. Introduction........................................................................................................7 1.1. The Genus Allium .................................................................................................7 1.1.1. History of the Genus Allium ..................................................................................7 1.1.2. Botany and Taxonomy of the Genus Allium .........................................................9 1.1.3. Sulphur Compounds of Allium ............................................................................11 1.1.4. Non-Sulphur Compounds of Allium ....................................................................13 1.1.5. Enzymes of Allium ..............................................................................................14 1.1.6. Bioactivity and Medicinal Use of Garlic (Allium sativum L.) ................................17 1.1.7. Bioactivity and Medicinal Use of Onion (Allium cepa L.) ....................................17 1.1.8. Bioactivity and Medicinal Use of Further Onions (Allium spec.) .........................18 1.2. Analytical Methods for the Determination of Allium Compounds........................18 1.2.1. Thin layer Chromatography (TLC) ......................................................................19 1.2.2. Low Pressure Liquid Chromatography (LC) .......................................................19 1.2.3. High Performance Liquid Chromatography (HPLC) ...........................................20 1.2.4. Flow-Through and Flow-Injection Analysis (FIA) ................................................20 2. Scope of Investigation ....................................................................................23 2.1. Screening on Cysteine Sulphoxides ...................................................................23 2.2. Testing for Bioactivity..........................................................................................23 2.3. Aspects of Chemotaxonomy...............................................................................24 3. Material and Methods ......................................................................................25 3.1. List of Investigated Allium Species .....................................................................25 3.2. Reagents and Buffers.........................................................................................34 3.3. Equipment Used for Investigation.......................................................................40 3.4. LC-Separation.....................................................................................................41 3.5. TLC-Separation ..................................................................................................43 3.6. HPLC Separation................................................................................................44 3.6.1. Sample Preparation............................................................................................44 3.6.2. HPLC Method .....................................................................................................44 3.7. Flow Injection Analysis (FIA) ..............................................................................48 3.7.1. Sample Preparation............................................................................................48 3.7.2. FIA Method .........................................................................................................49 3.8. Enzyme Isolation and Partial Purification ...........................................................52 3.9. Isolation and Structure Elucidation of the Red Dye ............................................53 3.10. Isolation and Structure Elucidation of a Possible Precursor ...............................54 3.11. Antibiotic activity .................................................................................................54 3.11.1. Sample Preparation............................................................................................55 3.11.2. Agar Diffusion Test .............................................................................................56 3.12. Radical scavenger activity ..................................................................................59 3.12.1. Sample Preparation............................................................................................60 3.12.2. Determination of Radical Scavenger Activity using DPPH .................................62 4. Results..............................................................................................................65 4.1. Flow Injection Analysis (FIA) ..............................................................................65 4.1.1. FIA-System Development...................................................................................65 4.1.2. Calibration of FIA with Ammonium and Alliin......................................................67 4.1.3. FIA Substrate Specificity.....................................................................................69 4.1.4. Limits of Detection and Quantitation...................................................................73 4.1.5. Analysis of Allium Species by FIA ......................................................................74 4.2. HPLC Analysis....................................................................................................77 4.2.1. Analysis from Allium Species Collected in Iran...................................................77 4.2.2. Analysis from Allium Species Collected in Turkmenistan ...................................80 4.2.3. Analysis from Allium Species Collected in Uzbekistan .......................................83 1 List of Contents 4.2.4. Analysis from Allium Species Collected in Tajikistan..........................................88 4.2.5. Analysis from Allium Species from the IPK-Collection, Gatersleben ................104 4.2.6. Comparison Between HPLC and FIA ...............................................................109 4.3. Structure Elucidationof the Red Dye.................................................................110 4.4. Antibiotic Activity of Allium Extracts ..................................................................111 4.4.1. Pre-Screening on Antibacterial, Antifungal and Antialgal Activity.....................111 4.4.2. Broad Screening on Antibacterial Activity (Experiment No. 1)..........................115 4.4.3. Selective Screening on Antibacterial Activity (Experiment No. 2).....................117 4.5. Radical Scavenger Activity of Allium Extracts ..................................................127 5. Discussion......................................................................................................133 5.1. Methodology .....................................................................................................133 5.1.1. HPLC-Method...................................................................................................133 5.1.2. FIA-Method .......................................................................................................133 5.2. Intraspecific Comparison of Cysteine Sulphoxides and Possible Pharmaceutical Use ..................................................................................................................134 5.2.1. Allium barsczewskii...........................................................................................135 5.2.2. Allium oschaninii...............................................................................................137 5.2.3. Allium paradoxum var. normale ........................................................................138 5.2.4. Allium caspium..................................................................................................140 5.2.5. Allium komarowii...............................................................................................141 5.2.6. Allium giganteum ..............................................................................................144 5.2.7. Allium pskemense.............................................................................................146 5.2.8. Allium suworowii ...............................................................................................148 5.2.9. Allium jesdianum...............................................................................................149 5.2.10. Allium aflatunense ............................................................................................151 5.2.11. Allium filidens....................................................................................................152