Determination of Α- Glucosidase Inhibitory Activity and Phytochemical Investigation of Bauhinia Malabarica
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Determination of α- glucosidase inhibitory activity and phytochemical investigation of Bauhinia malabarica Diploma thesis Submitted to the Faculty of Natural Sciences Karl- Franzens- University Graz For the degree “Magistra Pharmaciae” Presented by Elisabeth Plhak Graz, December 2015 “Das Wissen ist in unserem Leben, was die Blume im nützlichen Gras ist: sie gibt ihren Duft zum Futter.” Ludwig Ganghofer, “Der Hohe Schein“ 2 This research work was accomplished at the Department of Pharmacognosy and Pharmaceutical Botany Faculty of Pharmaceutical Sciences Prince of Songkla University Hat Yai Thailand and the Institute of Pharmaceutical Sciences Department of Pharmacognosy Faculty of Natural Sciences Karl- Franzens- University Graz Austria 3 Acknowledgements First of all I want to thank Prof. Dr. Sukanya Dej- adisai for supporting me at my work and for answering all my questions. Furthermore I am grateful to her for the organisation of my stay in Thailand and the excursions I was able to join with her and her students. I want to thank Prof. Dr. Adelheid Brantner for supervising me at the University of Graz and for making my stay in Thailand possible. Furthermore I am very thankful for her useful advices concerning cultural aspects. I want to thank the Phd- and Master- students in the laboratory at the Institute of Pharmacognosy in Hat Yai, especially Thanet Pitakbut, Wanlapa Nuankaew and Sathianpong Klaewchit for introducing me to the working techniques in the laboratory and sharing their knowledge, experience and free time with me. I am grateful for the time we spent together not only in the laboratory but also at the holidays and the family celebrations. For my work at the University of Graz I want to thank Dr. Sara Crockett, Ing. Elvira Knauder and Dr. Volker Wolkinger for introducing me to work with analytical and semi preparative HPLC. I want to thank Ass.- Prof. Dr. Olaf Kunert for making the NMR spectra of my fractions and helping me with the structure elucidation. Furthermore I want to thank Prof. Dr. Bernhard-Michael Mayer for the provision of the microplate reader and Heike Stessel for showing me how to work with it. I want to thank all my friends, for spending their time with me and helping me to get my head off. Special thanks to my dear colleague Sabine Ittensammer- Prinz for the time we spent together during our studies and in Thailand. I am so grateful to call her my friend and for every experience we had together. Finally I want to thank Ingeborg Plhak, Johann Plhak, Alexander Plhak and Maria Graf for their great mental and financial support and for motivating and encouraging me during my studies. 4 Table of contents Acknowledgements .......................................................................................................... 4 1. Introduction .............................................................................................................. 8 2. Objectives ................................................................................................................ 10 THEORETICAL PART ................................................................................................... 11 3. The Fabacea family ................................................................................................ 12 3.1. The subfamily of Caesalpinoideae and the genus Bauhinia ........................ 12 3.2. The genus Bauhinia ......................................................................................... 13 4. Bauhinia malabarica ............................................................................................... 13 4.1. Botanical classification .................................................................................... 14 4.2. Botanical description of Bauhinia malabarica .............................................. 15 4.3. Distribution ...................................................................................................... 16 4.4. Ecology ............................................................................................................. 16 4.5. Medical use....................................................................................................... 16 4.6. Phytochemical studies ..................................................................................... 17 EXPERIMENTAL PART ................................................................................................. 21 5. Processing of the raw plant material .................................................................... 22 6. Percolation of Bauhinia malabarica leaves ........................................................... 22 6.1. Preparation of the plant extracts ................................................................... 24 6.1.1. Hexane extract ...................................................................................................... 24 6.1.2. Ethyl acetate extract ............................................................................................. 24 6.1.3. Ethanol extract ..................................................................................................... 25 6.1.4. Water extract ........................................................................................................ 26 7. Methods used for the isolation of natural products ............................................ 27 7.1. Theory of thin layer chromatography (TLC) ............................................... 27 7.1.1. Preparative thin- layer chromatography (PTLC) ................................................ 27 7.2. Theory of low pressure liquid column chromatography (LPLC) ............... 28 7.2.1. Adsorption LPLC using silica gel as stationary phase ........................................ 28 7.2.2. Size exclusion LPLC using Sephadex® LH- 20 as stationary phase ................... 29 7.3. Liquid- liquid extraction (partitioning)......................................................... 30 7.4. High- performance liquid chromatography (HPLC) ................................... 30 7.4.1. Detection methods ................................................................................................ 30 8. TLC- Screening ....................................................................................................... 32 5 8.1. Spraying reagents ............................................................................................ 32 8.1.1 Dragendorff reagent (DRG) ................................................................................. 32 8.1.2. Vanillin- sulfuric acid reagent ............................................................................. 32 8.1.3. Iron- III- chloride reagent .................................................................................... 32 8.1.4. Fast blue salt reagent (FBS) ................................................................................ 32 8.1.5. Natural products- polyethylene glycol reagent (NP/ PEG) ................................. 33 8.2. Screening on alkaloids .................................................................................... 34 8.3. Screening on terpenes and essential oils ........................................................ 36 8.4. Screening on tannins ....................................................................................... 38 8.5. Screening on flavonoids .................................................................................. 40 8.6. Results and interpretation of the TLC screening ......................................... 41 9. Investigations of the hexane extract ...................................................................... 42 9.1. Flash chromatography on a silica gel column .............................................. 42 9.2. Silica gel column 1 ........................................................................................... 44 9.3. Silica gel column 2 ........................................................................................... 46 9.4. Liquid- liquid extraction ................................................................................. 47 9.5. Sephadex® LH- 20 column 1 .......................................................................... 48 9.6. Sephadex® LH- 20 column 2 .......................................................................... 49 9.7. PTLC ................................................................................................................ 49 10. Investigations of the ethanol extract ................................................................. 52 10.1. TLC- screening of the EtOH extract.......................................................... 52 10.2. Liquid- liquid extraction by using an Extrelut®-column ........................ 54 10.2.1. CHCl3 fraction ...................................................................................................... 54 10.2.2. EtOAc fraction...................................................................................................... 55 10.2.3. n- BuOH fraction .................................................................................................. 56 10.3. HPLC- screening of the fractions ............................................................... 57 10.3.1. Analytical HPLC method 1 .................................................................................. 57 10.3.2. Analytical HPLC method 2 .................................................................................. 59 10.3.3. Analytical HPLC method 3 .................................................................................