Investigations on Wild Allium Species. Part I: Cysteine Sulfoxides of Flowers. Part 2: Anticancer Activity of Bulb Extracts Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Pharmazie der Philipps-Universität Marburg vorgelegt von Emil Jivishov aus Baku, Aserbaidschan Marburg/Lahn Jahr 2018 Erstgutachter: Prof. Dr. Michael Keusgen Zweitgutachter: Prof. Dr. Shuming Li Eingereicht am ………................. Tag der mündlichen Prüfung am 21.12.2016 Hochschulkennziffer: 1180 Dedicated to my wife and life partner Sevda, our little hearts- children Said and Esma, my beloved parents and my proud brother Orkhan, to the supporting parents-in-law and brother- in-law Pervin. If learning the truth is the scientist’s goal… then he must make himself the enemy of all that he reads Ibn al-Haytham (Alhazen) Table of contents Table of contents ................................................................................................................ 1 Acknowledgments .............................................................................................................. 4 List of figures ...................................................................................................................... 6 List of tables ....................................................................................................................... 9 List of abbreviations ..........................................................................................................11 Summary ...........................................................................................................................14 Zusammenfassung ............................................................................................................15 1. INTRODUCTION .......................................................................................................17 1.1. The genus Allium ...................................................................................................17 1.2. General chemistry and sulfur compounds of Allium ................................................18 1.3. Role of arginine in Alliums and plants in general ....................................................24 1.4. Alliums and their anticancer activity ........................................................................27 1.4.1. Effect of sulfides from Allium spp on Nrf2/HO-1 antioxidant system in cancer cells…........................................................................................................................29 1.4.2. Sulfides from Allium spp as inducers of cell cycle inhibitor CDKN1a (p21) protein .......................................................................................................................30 1.5. Objectives of this investigation ...............................................................................31 1.5.1. Screening flowers of Allium species for cysteine sulfoxide and amino acids…………………………………………………………………………………………..31 1.5.2. Anticancer effect of bulbs of Allium species ....................................................32 2. MATERIALS AND METHODS ...................................................................................34 2.1. Plant materials .......................................................................................................34 2.1.1. Preparation of ethyl acetate extracts of plant material .....................................41 2.2. Reagents, equipment and devices .........................................................................43 2.2.1. Preparation of borate buffer pH 9.5 .................................................................45 2.2.2. Preparation of derivatisation agent ..................................................................45 2.2.3. Preparation of 1M Iodoacetamide solution ......................................................45 2.2.4. Preparation of phosphate buffer (pH 6.5) for HPLC .........................................45 2.2.5. Preparation of 50 mM ammonium acetate buffer (pH 6.5) for HPLC-MS .........46 2.3. HPLC preparations and analyses ...........................................................................46 2.3.1. Quantitative and qualitative analyses of Allium flower extracts derivatized with OPA ……………………………………………………………………………………...... 46 1 2.3.2. Bioactivity guided fractionation of A. aflatunense, A. stipitatum, A. rosenorum and A. pallens ............................................................................................................49 2.4. Bioactivity tests ......................................................................................................51 2.4.1. Human bladder cancer cell lines (T24 and UMUC3) and human fibroblast cell line (HFF-1) ...............................................................................................................53 2.4.2. XTT cell proliferation assay .............................................................................53 2.4.3. Flow cytometry analysis of cell death in T24 cells treated with EtOAc extracts of A. aflatunense, A. stipitatum, A. rosenorum and A. pallens ........................................53 2.4.4. RNA and protein analytics ...............................................................................54 3. RESULTS ..................................................................................................................56 3.1. Quantitative and qualitative HPLC analysis of CSOs and amino acids in flowers of Allium spp. .....................................................................................................................56 3.1.1. Total CSO content in samples .........................................................................56 3.1.2. Total amino acid content in samples ...............................................................57 3.2. Bioactivity against cancer cells ...............................................................................62 3.2.1. Inhibition of cell proliferation by Allium EtOAc extracts in T24 and UMUC3 cells …………………………………………………………………………………………62 3.2.2. Flow cytometry assay results of EtOAc extracts of Allium spp. ........................67 3.2.3. Allium extracts upregulates Nrf2, HO-1, and CDKN1A mRNA .........................68 3.3. Bioactivity guided fractionation of four most cytotoxic Allium spp EtOAc extracts ...70 3.4. Fractions from A. stipitatum induce expression of hemeoxygenase-1 (HO-1) in T24 cells. ..............................................................................................................................73 3.5. Amino acid and CSO content of 7002 A. stipitatum and 1178 A. aflatunense bulbs…………. ..............................................................................................................75 3.6. Structure elucidation of some fractions from A. stipitatum and A. aflatunense ........76 4. DISCUSSION ............................................................................................................78 4.1. Essential amino acid composition of flowers from subgenus Melanocrommyum ....78 4.1.1. Allium rosenorum ............................................................................................78 4.1.2. Section Compactoprason ................................................................................80 4.1.3. Section Acanthoprason ...................................................................................81 4.1.4. Section Kaloprason .........................................................................................81 4.1.5. Section Megaloprason ....................................................................................82 4.1.6. Section Melanocrommyum ..............................................................................83 2 4.1.7. Section Popovia ..............................................................................................85 4.1.8. Section Procerallium .......................................................................................85 4.2. Cysteine sulfoxide composition of flowers from subgenus Melanocrommyum ........87 4.2.1. Methiin ............................................................................................................87 4.2.2. Allium rosenorum ............................................................................................88 4.2.3. Section Compactoprason ................................................................................90 4.2.4. Section Acanthoprason ...................................................................................91 4.2.5. Section Kaloprason .........................................................................................92 4.2.6. Section Megaloprason ....................................................................................93 4.2.7. Section Melanocrommyum ..............................................................................94 4.2.8. Section Popovia ..............................................................................................95 4.2.9. Section Procerallium .......................................................................................95 4.3. Anticancer activity of Allium species .......................................................................98 5. CONCLUSION ............................................................................................................ 103 6. REFERENCES ............................................................................................................ 106 7.