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PREVENTION of UROLITHIASIS by Ammi Visnaga L

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PREVENTION of UROLITHIASIS by Ammi Visnaga L Ammi visnaga L. FOR THE PREVENTION OF UROLITHIASIS By PATTARAPORN VANACHAYANGKUL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 by Pattaraporn Vanachayangkul 2 To my family 3 ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Veronika Butterweck, for providing the greatest opportunity to work with her and support me in everyway in my studies here. I have had so many rewarding experiences, not only in academics but also in life. I would like to thank Dr. Saeed Khan who guided me throughout my research of nephrolithiasis all of the experimentation performed (kidney stone disease) and all of the experiment that I have done in his laboratory. Also, I would like to thank my dissertation committee members, Dr. Hartmut Derendorf and Dr. Cary Mobley for their valuable recommendations. I am grateful for their help and advice from Dr. Guenther Hochhaus and Dr. Jeffry Hughes. Special thanks go to the research groups of Dr. Saeed Khan, Karen Byer who assisted and guided me in cell culture experimentation and also Pat Glenton who helped me during my animal experimentation. I am in appreciation of the help from Dr. Reginald Frye in developing and working with the LC/MS method for my project. Also, I would like to thank Dr. Karin Woelkart for her advice and experience in animal experimentation. I would also like to thank Sasiporn Sarawek and Witcha Imaram for true friendship and a very generous support in every manner since the first day I arrived. I am grateful and thankful of my lab members and friends who were the biggest support for me throughout my study. Victor, Oliver, Navin, Matt, Immo, Anna-Maria, Nick, Lydia and Ane have been a great help in being there for professional and personal advice. Finally, I would like to thank my family for supporting, understanding and being there with love, care and encouraging words. The person that I would like to thanks the most is my father. Without him, my life would not turn out the way I did. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .......................................................................................................................10 LIST OF ABBREVIATIONS........................................................................................................12 ABSTRACT...................................................................................................................................13 CHAPTER 1 INTRODUCTION ..................................................................................................................15 Urolothiasis : Kidney Stone....................................................................................................15 Kidney Function ..............................................................................................................16 Initiation of Calcium Oxalate Stone in Renal Epithelial Cells........................................16 Animal Model..................................................................................................................18 Risk Factor of Kidney Stone Disease..............................................................................18 Treatment and Prevention of Kidney Stone Disease.......................................................19 Folk Medicine for Treatment of Urolithiasis...................................................................20 Ethnopharmacology of Ammi visnaga L. (Khella).................................................................20 Pharmacokinetics....................................................................................................................22 Hypothesis and Specific Aims................................................................................................24 2 IDENTIFICATION AND QUANTIFICATION OF COMPOUNDS IN KHELL EXTRACT..............................................................................................................................33 Background.............................................................................................................................33 Specific Aim ...........................................................................................................................33 Materials and Method.............................................................................................................33 Chemicals ........................................................................................................................33 Plant Material ..................................................................................................................33 Thin Layer Chromatography Analysis ............................................................................34 High Performance Liquid Chromatography with Diode Array Detector (HPLC/DAD) Analysis................................................................................................34 Sample preparation for HPLC analysis....................................................................34 Work solution and the preparation of Calibration Standards...................................35 Quantification...........................................................................................................35 Validation.................................................................................................................35 Results.....................................................................................................................................36 Thin Layer Chromatograohy Analysis ............................................................................36 High Performance Liquid Chromatography Analysis.....................................................36 Discussion and Conclusion.....................................................................................................37 5 3 EVALUATE THE PREVENTIVE EFFECT OF KEHLLA EXTRACTS ON OXALATE AND CALCIUM OXALATE CRYSTALS EXPOSED TO IN VITRO CELL CULTURE...................................................................................................................45 Background.............................................................................................................................45 Specific Aim ...........................................................................................................................45 Materials and Methods ...........................................................................................................45 Cell lines, Chemicals and Biochemicals .........................................................................45 Plant Material ..................................................................................................................46 Extraction procedure ................................................................................................46 Sample preparation...................................................................................................46 Cell Culture Experiment..................................................................................................46 Dose response experiment of Khella........................................................................47 Pure compound experiment......................................................................................47 Lactate dehydrogenase (LDH) Assay ......................................................................47 Statistical analysis ....................................................................................................48 Results.....................................................................................................................................48 Dose Response Experiment of Khella.............................................................................48 Pure Compound Experiment ...........................................................................................49 Discussion and Conclusion.....................................................................................................49 4 EVALUATE THE PREVENTION OF HYPEROXAURIA BY AN EXTRACT OF Ammi visnaga L. AND MAJOR COMPOUNDS IN STONE FORMING RATS.................58 Background.............................................................................................................................58 Specific Aim ...........................................................................................................................58 Materials and Methods ...........................................................................................................59 Chemicals and Biochemicals...........................................................................................59 Plant Material and Extraction Procedure.........................................................................59 Sample preparation...................................................................................................59 Animals and Experimental Protocols ..............................................................................59 Oxalate, citric acid and calcium determination........................................................60
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