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Ammi visnaga L. for the Prevention of Urolithiasis

Permanent Link: http://ufdc.ufl.edu/UFE0022856/00001

Material Information

Title: Ammi visnaga L. for the Prevention of Urolithiasis
Physical Description: 1 online resource (116 p.)
Language: english
Creator: Vanachayangkul, Pattaraporn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ammi, khella, kidney, pharmacokinetic, stone, urolithiasis, visnaga
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Urolithiasis is a clinical condition referred to as kidney stone disease. This disease is common and currently may affect many people in industrialized countries. Several factors can promote the formation of kidney stones such as dehydration, consumption of certain foods that contain high amount of calcium, oxalate or uric acid and some infectious diseases. Most people can develop kidney stones sometime in their lives and may pass stones through the urinary tract unnoticed. The stones usually composed mainly of calcium oxalate (CaOx). However, if the stone enlarges, it will obstruct the urinary system. Symptoms initially present with severe pain and if the stone damages the urinary tube, then blood can be excreted in the urine. The most effective treatment of kidney stones is extracorporeal shock wave lithotripsy (ESWL), but this treatment still has several disadvantages including high costs, the reoccurance of kidney stones and potential renal damage. Therefore, alternative medication with less side effects it is of great interest to investigate. Ammi visnaga L. or Khella (KE, Apiaceae) is traditionally used as treatment for kidney stones as a tea preparation from Egypt. KE is used to relieve the pain and help the stone pass through the ureter. On the basis of this consideration, KE was characterized and investigated for the preventive effect of kidney stone formation in our study. HPLC analysis was used to identify and quantify KE. In cell culture experiments, it was found that KE and its compounds protect cell damage from CaOx crystals. In addition, KE and its compounds prevented CaOx formation in stone forming rats by increasing the urinary pH and citrate concentration along with a decrease of urinary oxalate. The CaOx crystals deposition in the rat kidneys was significantly decreased in the group of rats receiving KE and its compounds. The pharmacokinetic study of visnagin was investigated and revealed that visnagin was a short half-life compound with complete absorption. In conclusion, results indicated that KE could be used as a preventive agent for kidney stone disease.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Pattaraporn Vanachayangkul.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Butterweck, Veronika.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022856:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022856/00001

Material Information

Title: Ammi visnaga L. for the Prevention of Urolithiasis
Physical Description: 1 online resource (116 p.)
Language: english
Creator: Vanachayangkul, Pattaraporn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ammi, khella, kidney, pharmacokinetic, stone, urolithiasis, visnaga
Pharmacy -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Urolithiasis is a clinical condition referred to as kidney stone disease. This disease is common and currently may affect many people in industrialized countries. Several factors can promote the formation of kidney stones such as dehydration, consumption of certain foods that contain high amount of calcium, oxalate or uric acid and some infectious diseases. Most people can develop kidney stones sometime in their lives and may pass stones through the urinary tract unnoticed. The stones usually composed mainly of calcium oxalate (CaOx). However, if the stone enlarges, it will obstruct the urinary system. Symptoms initially present with severe pain and if the stone damages the urinary tube, then blood can be excreted in the urine. The most effective treatment of kidney stones is extracorporeal shock wave lithotripsy (ESWL), but this treatment still has several disadvantages including high costs, the reoccurance of kidney stones and potential renal damage. Therefore, alternative medication with less side effects it is of great interest to investigate. Ammi visnaga L. or Khella (KE, Apiaceae) is traditionally used as treatment for kidney stones as a tea preparation from Egypt. KE is used to relieve the pain and help the stone pass through the ureter. On the basis of this consideration, KE was characterized and investigated for the preventive effect of kidney stone formation in our study. HPLC analysis was used to identify and quantify KE. In cell culture experiments, it was found that KE and its compounds protect cell damage from CaOx crystals. In addition, KE and its compounds prevented CaOx formation in stone forming rats by increasing the urinary pH and citrate concentration along with a decrease of urinary oxalate. The CaOx crystals deposition in the rat kidneys was significantly decreased in the group of rats receiving KE and its compounds. The pharmacokinetic study of visnagin was investigated and revealed that visnagin was a short half-life compound with complete absorption. In conclusion, results indicated that KE could be used as a preventive agent for kidney stone disease.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Pattaraporn Vanachayangkul.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Butterweck, Veronika.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022856:00001


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1 Ammi visnaga L. FOR THE PREVENTION OF UROLITHIASIS By PATTARAPORN VANACHAYANGKUL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 by Pattaraporn Vanachayangkul

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3 To my family

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4 ACKNOWLEDGMENTS I would like to thank m y advisor, Dr. Ver onika 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 e xperiment that I have done in his laboratory. Also, I would like to thank my dissertation co mmittee 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 By er 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 he lp from Dr. Reginald Frye in developing and working with the LC/MS method for my project. Also, I would like to th ank 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 firs t day I arrived. I am grat eful and thankful of my lab members and friends who were the bi ggest support for me throughout my study. Victor, Oliver, Navin, Matt, Immo, Anna-Maria, Nick, Lydi a 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 pers on that I would like to thanks the most is my father. Without him, my life woul d not turn out the way I did.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.......................................................................................................................10 LIST OF ABBREVIATIONS........................................................................................................ 12 ABSTRACT...................................................................................................................................13 CHAP TER 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 QUANTIFICAT ION OF COM POUNDS 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 Chromatogr aphy 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

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6 3 EVALUATE THE PREVENTIVE EFFE CT OF KEHL LA 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 HYPE R OXAURIA 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 Lactate dehydrogynase and al kaline phosphatase assay ..........................................60 Calcium oxalate crystal deposition in kidney .......................................................... 60 Statistical analysis.................................................................................................... 61 Results.....................................................................................................................................61 Khella Experiment...........................................................................................................61 Survival rate............................................................................................................. 61 Lactate dehydrogenase and alkaline phosphatase.................................................... 61 Urine volume Calcium, oxalate, citrate, and pH...................................................... 61 Calcium oxalate crystal depositions......................................................................... 62 Major Compound Experiment......................................................................................... 62 Discussion and Conclusion.....................................................................................................62

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7 5 PHARMACOKINETIC ANALYSIS OF VISNAGIN IN RATS ..........................................80 Background.............................................................................................................................80 Specific Aim...........................................................................................................................81 Materials and Methods...........................................................................................................81 Chemicals........................................................................................................................81 Liquid Chromatography/Tendem Mass Spectrom etry (LC-MS/MS)Analysis............... 81 Stock, work solution, and prepara tion of calibrati on standards ............................... 81 LC-MS/MS condition...............................................................................................82 Method validation....................................................................................................83 Animals Experiment........................................................................................................ 84 Analytical Methods......................................................................................................... 85 Sample preparation...................................................................................................85 Statistical analysis.................................................................................................... 85 Results.....................................................................................................................................85 Method validation............................................................................................................85 Pharmacokinetic Study of Visnagin................................................................................ 86 Discussion and Conclusion.....................................................................................................87 Method Validation...........................................................................................................87 Pharmacokinetic Study of Visnagin................................................................................ 87 6 CONCLUSION..................................................................................................................... ..97 ADDITIONAL TABLES...............................................................................................................99 Cell Culture Experiment.........................................................................................................99 Animal Experiment...............................................................................................................102 REFERENCES............................................................................................................................107 BIOGRAPHICAL SKETCH.......................................................................................................116

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8 LIST OF TABLES Table page 1-1 Salient features of CaOx nephrolithia sis in hum ans and chronically but mildly hyperoxaluric rats (15-17).................................................................................................. 26 1-2 Effect of Ammi visnga L. (500 m g/kg) in urine output (43)..............................................27 1-3 Effect of Ammi visnaga L. on calcium and oxalate contents of kidney of rats fed with 3% glycol acid for 4 week (43) ..........................................................................................27 2-1 Concentrations of the standard solutions used for the calibration curves and quality controls (Q Cs) of khellin and visnagin.............................................................................. 39 2-2 Intra-day (n=3) and inter-day (n=9) a ssay parameters of khellin and visnagin. Precis ion expressed as %CV and accuracy expressed as % of the theoretical concentration......................................................................................................................39 2-3 Stability test of khellin and visnagin af ter 24 hours on autosampler at 20 C. Data represents the percen tage remaining of all test compounds............................................... 40 2-4 Quantitative analysis of khellin and vinagi n in KE (water ex tract). Data represents the mean SD mg/g freeze-dried extract.......................................................................... 40 3-1 Dose regimen for dose response experiment of KE in cell culture.................................... 52 3-2 Dose regimen for pure compound experiment................................................................... 53 4-1 Dose regimen of KE...........................................................................................................66 4-2 Dose regimen of khellin and visnagin............................................................................... 66 5-1 concentrations of working solution us ed for preparing the standard solution ...................90 5-2 Concentrations of standard solution us ed for the calibration curves and quality control ................................................................................................................................91 5-3 Intra-day (n=8) and inter-day (n=24) precis ion (%CV) and accuracy (%RE) for analysis of visnagin in blank rat plasma............................................................................ 92 5-4 Pharmacokinetic parameters from mean plasm a conetration-time profile using noncompartmental analysis after oral and intravenous administration (n=8).................... 93 A-1 The % LDH release from oxalate exposure to L LC-PK1 with or without KE (N=6).......99 A-2 The % LDH release from calcium oxalat e crystals exposure to LLC-PK1 with or without KE (N=6) ..............................................................................................................99

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9 A-3 The % LDH release from oxalate exposur e to MDCK with or without K E (N=6)........... 99 A-4 The % LDH release from calcium oxalat e crystals exposure to MDCK wi th or without KE(N=6).............................................................................................................100 A-5 The % LDH release from oxalate exposure to L LC-PK1 with or without khellin or visnagin (N=6)................................................................................................................. 100 A-6 The % LDH release from calcium oxalat e crystals exposure to LLC-PK1 with or without khellin or visnagin (N=6) ...................................................................................100 A-7 The % LDH release from oxalate exposur e to MDCK with or without khellin or visnagin (N=6) ................................................................................................................. 101 A-8 The % LDH release from calcium oxalat e crystals exposure to MDCK wi th or without khellin or visnagin (N=6)...................................................................................101 A-9 Lactate dehydrogenase (LDH)and alkaline phosphatase (A LP) in urine (U/L) at day 0 after treatm ent with KE................................................................................................. 102 A-10 Lactate dehydrogenase (LDH) and alkaline phosphatase (A LP) in urine (U/L) at day 7 after treatm ent with KE................................................................................................. 102 A-11 Lactate dehydrogenase (LDH)and alkaline phosphatase (A LP) in urine (U/L) at day 14 after treatm ent with KE............................................................................................... 102 A-12 Calcium (Ca), citrate, oxalate (Ox) in urine (U/L ) at da y 0 after treatment with KE...... 103 A-13 Calcium (Ca), citrate, oxalate (Ox) in urine (U/L ) at da y 7 after treatment with KE...... 103 A-14 Calcium (Ca), citrate, oxalate (Ox) in urine (U/L ) at da y 14 after treatment with KE....104 A-15 Calcium oxalate crystal de position and score in kidney ..................................................104 A-16 Calcium (Ca), citrate, oxalate (Ox) in urine (U/L ) at day 0 after treatment with khellin or visnagin............................................................................................................105 A-17 Calcium (Ca), citrate, oxalate (Ox) in urine (U/L ) at day 7 after treatment with khellin or visnagin............................................................................................................105 A-18 Calcium (Ca), citrate, oxalate (Ox) in urine (U/L ) at day 14 after treatment with khellin or visnagin............................................................................................................106 A-19 Calcium oxalate crystal deposition and scor e in kidney after treatm ent with khellin or visnagin....................................................................................................................... .....106

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10 LIST OF FIGURES Figure page 1-1 Structure of the urinary system (4).................................................................................... 28 1-2 Normal nephron (7)............................................................................................................29 1-3 Oxalate action on renal cells (8)........................................................................................ 30 1-4 Schematic presentation of relationships be tween various factors, which lead to the for mation of idiopathic kidney stone (8, 9)....................................................................... 31 1-5 Ammi visnaga L.: Flowering tops, Helsinki Botanical garden, Finland ............................ 32 1-6 Main compounds from Khella........................................................................................... 32 2-1 Representation of Thin Layer Chromatography plate result of Ammi visnaga L. and Ammi majus L. ...................................................................................................................41 2-2 An HPLC chromagram......................................................................................................42 2-3 Mean Calibration curves of com pounds in Khella extrac t (n=9). A) Khellin and B) Visnagin. Vertical bars represent the st andard deviations (SD) of the means................... 43 2-4 Absorbance-wavelength spectras....................................................................................... 44 3-1 The % LDH release from oxalate or calci um oxalate crystals exposure to LLC-PK1 with or without KE (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001).................................................................................................................................54 3-2 The % LDH release from oxalate or cal cium oxalate crystals exposure to MDCK with or without KE (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001).................................................................................................................................55 3-3 The % LDH release from oxalate or calci um oxalate crystals exposure to LLC-PK1 with or without pure com pounds (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001)............................................................................................................56 3-4 The % LDH release from oxalate or cal cium oxalate crystals exposure to MDCK with or without pure com pounds (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001)............................................................................................................57 4-1 Survival rate of animal after treatment with KE................................................................ 67 4-2 Lactate dehydrogenase in urine at day 7 (U/L) after treatm ent with KE........................... 68 4-3 Alkaline phosphatase in urine at da y 7 (U/L) after treatm ent with KE............................. 68

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11 4-4 Urine volume at day 7 (mL) after treatment with KE........................................................ 69 4-5 Urinary calcium excretion at day 7 (mg/d) after treatm ent with KE................................. 69 4-6 Urinary oxalate excretion at day 7 (mg/d) after treatm ent with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001)....................................... 70 4-7 Urinary citrate excretion at day 7 (mg/ d) after treatm ent with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001)....................................... 70 4-8 Urine pH after treatment with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001)..................................................................................................71 4-9 Calcium oxalate crystal deposition scor e, <1= 0, <10 = 1, <30 = 2, <50 = 3, < 75 = 4 and 75 = 5 after treatm ent with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001)...........................................................................................71 4-10 Histopathology of rat kidney (arrows point ed at the calcium oxalate crystals)................. 72 4-11 Urinary calcium excretion (mg/d) at day 7 and day 14 after treatm ent with khellin and visnagin (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).................................................................................................................................73 4-12 Urinary oxalate excretion (mg/d) at day 7 and day 14 after treatm e nt with khellin and visnagin (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).................................................................................................................................74 4-13 Urinary citrate excretion (mg/d) at day 7 and day 14 after treatm e nt with khellin and visnagin (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).................................................................................................................................75 4-14 Urinary pH excretion (mg/ d) at day 7 and day 14 after treatm ent with khellin and visnagin (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).................................................................................................................................76 4-15 Calcium oxalate crystal deposition scor e, <1= 0, <10 = 1, <30 = 2, <50 = 3, < 75 = 4 and 75 = 5 after treatm ent with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001)...........................................................................................77 4-16 Handling of citrate by hypot hetical renal tubule(81). ........................................................ 78 4-17 Potential mechanism of increased urinar y citrate with system ic alkalosis(82)................. 79 5-1 Structures of (A) visnagin and (B) warfarin (internal standard). ....................................... 94 5-2 The extracted LC-MS/MS chromatograms........................................................................ 95 5-3 Plasma concentration-time curve....................................................................................... 96

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12 LIST OF ABBREVIATIONS ACN Acetronitr ile ALP Alkaline phosphatase AM Acclimization Media CaOx Calcium oxalate COM Calcium oxalate monohydrate DAD Diode array detector EG Ethylene glycol ESWL Extracorporeal shock wave lithrotripsy HPLC High performance liquid chromatography KE Khella extract LC-MS/MS Liquid Chromatography /tandem mass spectrometry LDH Lactate dehydrogenase LLC-PK1 Porcine kidney proxima l tubular epithelial cell line MDCK Madin-Canine Kidney collecti ng duct tubular epithelium cell line NH4Cl Ammonium chloride Ox Oxalate PCi Potassium citrate TLC Thin layer chromatography

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13 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Ammi visnaga L. FOR THE PREVENTION OF UROLITHIASIS By Pattaraporn Vanachayangkul December 2008 Chair: Name Veronika Butterweck Major: Pharmaceutical Sciences Urolithiasis is a clinical condi tion referred to as kidney stone disease. This disease is common and currently may affect many people in industrialized countries Several factors can promote the formation of kidney stones such as dehydration, consumption of certain foods that contain high amount of calcium, oxalate or uric acid and some infectious diseases. Most people can develop kidney stones sometime in their liv es and may pass stones through the urinary tract unnoticed. The stones usually composed mainly of calcium oxalate (CaOx). However, if the stone enlarges, it will obstruct the urinary system. Symptoms initially present with severe pain and if the stone damages the urinary tube, then blood can be excreted in the urine. The most effective treatment of kidney stones is extracorporeal shock wave lithotripsy (ESWL), but this treatment still has several disadvantages including high costs, the reoccurance of kidney stones and potential renal damage. Theref ore, alternative medication with less side effects it is of great interest to investigate. Ammi visnaga L. or Khella (KE, Apiaceae) is trad itionally used as treatment for kidney stones as a tea preparation from Egypt. KE is us ed to relieve the pain and help the stone pass through the ureter. On the basis of this consideration, KE was char acterized and investigated for the preventive effect of kidney stone formation in our study. HPLC analysis was used to identify

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14 and quantify KE. In cell culture experiments, it was found that KE and its compounds protect cell damage from CaOx crystals. In addition, KE and its compounds prevented CaOx formation in stone forming rats by increasing the urinar y pH and citrate concentration along with a decrease of urinary oxalate. The CaOx crystals deposition in the rat kidneys was significantly decreased in the group of rats receiving KE and its compounds. The pharmacokinetic study of visnagin was investigated and revealed that visnagin was a short half-life compound with complete absorption. In conclusion, results indicate d that KE could be used as a preventive agent for kidney stone disease.

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15 CHAPTER 1 INTRODUCTION Urolothiasis : Kidney Stone Urolith aisis or nephrolithiasis represents th e clinical condition of kidney stone disease. Stone formation in the urinary tr act has been recognized for thousands of years, but during the last few decades the pattern and incidence of th e disease have changed markedly. About 5% of American women and 12% of men will develop a kidne y stone at some time in their life and the prevalence has been rising in both sexes (1). In the USA, over one million Americans each year are admitted to hospitals for the treatment of kidney stone disease. The reasons are multifold: life style, dietary habits and family history. Kidney stone formation (or urolithiasis) is a complex process that results from succession of se veral physico-chemical events including supersaturation, nucleation, growth, aggregation and retention within the renal tubules (2). Three distinct stages can be recogni zed in the process of stone fo rmation. The first stage involves crystal nucleation, growth and aggr egation. In the second stage, crys tals are retained within the kidneys, renal tubules and/or interstitium, and in the final stage, retained crystals move from inside of the kidney to the renal papillary surface to form a stone nidus. The latter grows into a stone by accretion of more crys tals. Epidemiological data have shown that calcium oxalate (CaOx) is the predominant mineral in the major ity of kidney stones (3). Initially, kidney stones often do not cause any symptoms. Usually, the first symptom of a kidney stone is extreme pain, which occurs when a stone acutely blocks the fl ow of urine. The pain often begins suddenly when a stone moves in the urinary tract, causing ir ritation or blockage. If the stone is too large and then moves, blood may appear in the urine.

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16 Kidney Function The urinary system is the organ system that produces, stores, and eliminates urine. In humans it includes two kidneys, two ureters, the bladder, and the urethra (Figure 1-1) (4). The kidneys lie behind the peritoneum at the back of the abdominal cavity. The kidney performs the essential function of removing waste products and maintaining body home ostasis. The renal cortex is the outer zone of the kidney and the renal medulla is the inner zone which is made up of the renal pyramids. The cortex contains all of the glomeruli and the medulla contains the loops of Henle, the vasa recta and the final portions of the collecting ducts. The nephron is the basic unit of the kidney, each kidney has 400,000-800,000 nephr ons, although this number declines with age (4-6). A nephron (Figure 1-2) co nsists of a glomerulus and an associated tubule that leads to the collecting duct. The urinary fi ltrate is formed in the glomerulus and passes into the tubules where the volume and content are altered by reab sorption or secretion. Most solute reabsorption occurs in the proximal tubules; whereas fine adju stments to urine composition are later made in the distal tubule and collecting duc ts. The loop of Henle serves to concentrate urine. In the proximal tubules, glucose, sodium chloride, and water are reabsorbed an d returned to the blood stream, along with essential nut rients such as amino acids, proteins, bicarbonate, calcium, phosphase, and potassium. In the loop of Henle, th e urine concentrating pr ocess proceeds and in the distal tubule the saltand acid -base balance of blood is regulate d. The final urine is formed in the collecting ducts, where the urine is drained into the calyces that leads to the ureter (4, 7). Initiation of Calcium Oxalate Stone in Renal Epithelial Cells In 2004, Julie A. Jonassen et al. summ arized the intracellular pathways activated by oxalate exposure that promote stone formati on, focusing on changes that enhance crystal attachment to renal cells and that provide cellula r debris for crystal nucleation (8), Figure 1-3. Supersaturation is the driving force behind crysta l formation of crystals which can be harmlessly

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17 expelled (9). Lethal epithelial cellu lar injury promotes crysta l nucleation, aggregation and retention, Figure 1-4. Urinary supers aturation controls cr ystallization but is it self controlled by the various consequences of crystal formation. Cr ystals that are not expelled with the urine induce production of crystalliza tion modulators such as glycoamionoglycan and various type of lipids and may eventually lead to cellular dysf unction and degradation. Products of cell injury promote further crystallization, from heteroge nous nucleation to crystal aggregation and retention. Cell injury also promot es interstitial inflammation, which is likely involved in crystal erosion to the papillary surface a nd the development of stone nidus. Although cell lines are not always a perf ect representation of cell behavior in vivo they form reliable models to repeatedly perform experiments under reproducible conditions. MardinDarby canine kidney collecting duct tubular ep ithelial cells (MDCK) and porcine kidney proximal tubular epithelial cells (LLC-PK1) are commonly used in kidney stone studies. MDCK was used as model systems for the distal pa rt of the nephron and c ontains many mammalian cortical collecting tubules (9, 10). MDCK cells display cell membrane enzymes -glutamyl transpeptidase (GGT) and leucine aminopeptid ase (LAP), the lysosomal enzyme N-acetylglucosaminidase (NAG), and the cytosolic enzyme lactate dehydrogenase. The absence of cilia was the feature most consisten tly associated with the cell ty pe thought to be analogous to collecting tubule intercalated ce lls (10, 11). The LLC-PK1 cell lin e possesses characteristics of the proximal tubule (such as Na+-dependent tran sport systems, enzymes located in the apical membrane including alkaline phosphase and -glutamyl transpeptidase). The LLC-PK1 cell line could be a relevant tool in th e examination of functions of pr oximal tubule cells (12, 13).

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18 Animal Model Calcium oxalate kidney stones in both humans and mildly hyperoxaluri a rats are located on renal papillary surfaces and consist of an organi c matrix and crystals of calcium oxalate(14). Male Sprague-Dawley rats were used in this study. The choice of these animals was made because this species is used frequently for studies of experimental nephrolithiasis. Stones formed in kidneys of humans and rats are identical and similarities between human and rats CaOx kidney stones are listed in Table 1-1 (15-17). Risk Factor of Kidney Stone Disease Several factors increase the risk for developi ng kidney stones, includ ing inadequate fluid intake and dehydration, reduced urinary flow and volum e, certain chemical levels in the urine that are too high (e.g. calcium, oxalate, uric acid) or too low (citrate), and several medical condition (16). Anything that bl ocks or reduces the flow of urine (e.g. urinary obstruction, genetic abnormality) also increases the risk. The modern western lifestyle provides a host factors that impair urine composition and thereby incr ease the risk of the stone formation. In our everyday life, we do not drink enough water and only twice or thri ce a day, we eat food that is too rich in calories and table salt, but have deficiencies in fiber a nd alkali. Last but not least, lack of exercise is not enough (18). The consequences are supersaturat ed urine and urine deficient in inhibitory substances, finally th e formation of stone occurred. Hyperoxaluria is a major risk factor of kidney stone disease (19). Oxalic acid is present in the foods such as chocolate, rhubarb or sweet potato and beverages, but ingested oxalate is poorly absorbed from the intes tine (20, 21). The exact etiology of increased urinary oxalate excretion remains to be elucidated. Increased di etary protein intake, alte red renal excretion and increased hepatic oxalate produc tion have all been postulate d as possible causes (20).

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19 Nonetheless, the preponderance of evidence sugges ts that increased intestinal absorption is the primary cause for hyperoxaluria in patients with calcium oxalate stone disease (20). Treatment and Prevention of Kidney Stone Disease A comm on and well-established treatment for the prevention of calcium stones is alkali citrate therapy (22). Potassium citrate is considered first-line treatment. Al kaline citrate increases the excretion of citrate mainly by increasing the pH of tubular cel ls (22). A small fraction of the absorbed citrate is excreted in the urine but the major part of administered citrate is metabolized. Thus, supersaturation with calcium oxalate is reduced and inhibi tion of growth and aggregation of corresponding crystal phases is increased. If the stone doe s not move through the ureter, surgery is considered. Thiazide is ideally indicated for the treatm ent of renal hypercalciuria. This diuretic has been shown to corr ect the renal leak of calcium by augmenting calcium reabsorption in proximal tubule and distal tubule (5). Oral ad ministration of large amount of calcium such as calcium citrate (0.25-1.0 g four times per day) has been recommended for the control of enteric hyperoxaluria by raising the urinary citrate and pH (5). Currentl y, the most effective treatment of kidney stone disease is extraco rporeal shock wave lithotrips y (ESWL) using highly focused impulses projected from outside the body to pulverize kidney st ones anywhere in the urinary system(22). The stone is usually reduced to sand-l ike granules that can be passed in the patients urine. Despite improvements to the procedure, its high cost and compelling data that now suggests exposure to shock wave in therapeutic doses may cause acute renal injury, decrease in renal function and an increase in stone recu rrence make this a undesireablbe treatment(23). Persistent residual stone fragme nts and the possibility of inf ection after ESWL represent a serious problem in the treatment of stones. A dditionally, even though drug treatment has shown some feasibility in many randomized trials, it is not accomplished without side effects(22).

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20 Therefore, it has been of great interest to dete rmine better alternative treatment options through herbal medicine. Folk Medicine for Trea tment o f Urolithiasis According to Saudi Arabian folk medicine, kidn ey stone patients are given teas made from seeds of Trigonella foenum-graecum (24). To understand the reason for the beneficial effects of this plant, CaOx nephrolithiasis was experime ntally induced in male rats by the dietary administration of 3% glycolic aci d. Daily oral treatment resulted in significant reduction in CaOx crystal deposition (24). The aerial part of Herniria hirsuta widely distributed in Mediterranean area, is used in folk medicine as a diuretic and to treat kidney stones (25).. An extract of H. hirsuta promoted the nucleation of calcium oxala te crystals, increasing their number but decreasing their size. It also pr omoted the formation of calcium oxalate dehydrate crystal, despite the presence of calcium oxalate monohydrate particles (25). Moreove r, to evaluate the prophylaxis effect, the rats were rendered nephrolythic by treati ng with oral administration of ethylene glycol 0.75% and ammonium chloride 1% for 3 days, the histology showed large deposits of CaOx crystal in all pa rts of the kidney in untreated rats but with almost no deposits in those of treated rats (26). Aqueous extract of another plant Phyllanthus niruri, used in Brazillian folk medicine for urolithiasis, was given to male rats with CaOx stone in the bladder and resulted in reduction of stone growth compared to control rats with no medicine (27). Ethnopharmacology of Ammi visn aga L. (Khella) Ammi visnaga L. (Khella, apiaceae) (Figure 1-5) is an annual Mediterranean herb, which grows wild from Morocco to the Near East, a nd has bi-or tripinatisect leaves with linear segments and white flowers grouped in large compound umbels (28). The fruits are ovateoblong (2-2.5 mm.) and can be distinguished from Ammi majus by their slightly protruding sides (29-31).

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21 People use Khella for several treatments. Ora lly, Khella is used for colic and abdominal cramps, kidney stones, menstrual pain, and premenstrual syndrome (PMS). Khella is also used for respiratory conditions including asthma, bronchitis, cough, and whooping cough. It is also used for cardiovascular disorder s including hypertension, cardiac arrhythmias, congestive heart failure (CHF), angina, atherosclerosis, and hyperchol esterolemia. It is also used for liver and gall bladder disorders, diabetes, and as a diuretic. Topically, Khella is used for vitiligo, psoriasis, wound healing, inflammation conditions, and poi sonous bites. There is some preliminary evidence that khellin might also increase high-de nsity lipoprotein (HDL) levels without affecting total cholesterol or triglyceride concentrations (32, 33). KE seems to have some antimicrobial activity. This might be attributable to both th e khellin and visnagin constituents, which both seem to have antifungal, antibacterial, and antivi ral activity (34). Research ers are interested in Khella for use in psoriasis. The khellin constituent is structurally similar to the psoralen nucleus and might be useful as a photosensitizer in patients with psoriasis (35). In traditional Egyptian medicine crushed or powdered fruits of Khella are used as tea, about 1 teaspoon of crushed fruits (approx. 20g per cup of hot water, infused for approximately 5 minutes (24). The main constituents are furanocoumarins (2-4%), including khellin (0.3-1.2%), visnagin (0.05-0.3%), khellol, and khellinol a nd angular pyrano-coumarin s (0.2-0.5%), including visnadin, samidin and dihydrosamidin. The t ea also contains lipids (up to 18%), furanoacetophenones, flavonoids (flavonol and flavonol sulfates), and 0.2-0.3mL/kg essential oil. The constituents, visnadin, visnag in, and khellin, all seem to have cardiovascular effects due to calcium channel blocking actions (33, 36). Visnadin is the most active, probably a result of its calcium blocking activity, shown in vitro (37). It can inhibit vascular smooth muscle contraction and seems to dilate peripheral and coronary vesse ls and increase coronary circulation (33, 36, 38-

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22 40). Visnagin also has negative chronotropic and inotropic effects and reduces peripheral vascular resistance (33, 41). Khel lin also acts as a vasodilator and has bronchodilatory activity and spasmolytic activity (36, 38, 42). Stuctures of the major compounds of Khella are shown in Figure 1-6. In 2001, Khan et al inve stigated the effect of Ammi visnaga seeds in animal experiment (43). Oxalate nephrolithiasis was induced by 3% glycolic acid given for 4 weeks. Daily oral treatment with Ammi visnaga (500mg/kg) highly reduced the incidence of calcium oxalate deposition in kidneys. In additi on, Khella seeds extract showed highly potent diuretic activity (Table 1-2). Moreover, the effect of Khella on calcium and oxa late contents in kidneys of rats fed with 3 % glycolic acid were studied (Table 1-3). Results revealed the effectiveness of Khella treatment on the inhibition of formation of calci um oxalate in kidneys. It was further supported by the observation that there were ve ry low (in some cases not at al l) deposition of calculi in the kidneys of Khella treated animals, as compared to the glycolic acid control group with heavy deposition in the form of patches. The prophylactic effect of Khella may possibly be attributed to its diuretic activity as can be seen from table 1 that the urine volume from treatment was higher than the normal group. Moreover, KE could inhi bit the formation of kidney stones by lowering the deposition of calculi in kidney. Pharmacokinetics Visnagin, one of the m ajor constituents in Ammi visnaga L., is a furanocoumarin derivative. Visnagin seems to have cardiovascular effects due to calcium channel blocking actions (33, 36). It can inhibit va scular smooth muscle contraction and seems to dilate peripheral and coronary vessels and increase coronary circulation (33, 36, 38-40). Visnagin also has negative chronotropic and inotropi c effects and reduces peripheral vascular resistance (33, 41).

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23 KE seems to have some antimicrobial activity. This might be attributable to both the khellin and visnagin constituents, which both seem to have antifungal, antibacterial, and antiviral activity (34). Although, visnagin have been studied in therapeutic use for over 10 years, its pharmacokinetic study has not b een investigated yet. Pharmacokinetic describes the quantitative relationship between administered doses, dosing regimens and (observed) plasma and/or tissue concentrations of the drugs (44). Pharmacokinetics is often studied in conjunc tion with pharmacodynamics. Pharmacodynamics explores what a drug does to the body, whereas pharmacokinetics explores what the body does to the drug. Pharmacodynamics studies the actions of drugs within the body; this includes the routes and mechanisms of absorption and excret ion, the rate at which a drug action begins and the duration of the effect, the bi otransformation of the substance in the body and the effects and routes of excretion of the metabolites of the drugs. Pharmacokinetics is divided into several ar eas which include the extent and rate of Absorption, Distribution, Metabo lism and Excretion. This commonly referred to as the ADME scheme (45, 46). Absorption is the process of a substance entering the body. Distribution is the dispersion or dissemination of substances th roughout the fluids and tissues of the body. Metabolism is the irreversible transformation of parent compo unds into daughter metabolites. Excretion is the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in a tissue in the body. Pharmacokinetics describes how the body aff ects a specific drug after administration. Pharmacokinetic properties of drugs may be af fected by elements such as the site of

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24 administration and the concentration in which th e drug is administered. These may affect the absorption rate (45, 46). Therefore, to obtain the information about absorption and disposition, the pharmacokinetics in rats trea ted with oral and in travenous administration of visnagin should be performed. Hypothesis and Specific Aims Kidney stones are the form ation of crystal aggr egates in the urinary tract. They affect 1012% of the population in industrialized countries (47). Approximately 2.4-bi llion dollars is spent annually to treat stone-related disorders. The stones may pass naturally, lodge elsewhere, or stay and grown in the kidney. The deposition of stones causes pain, blockage of the flow of urine, kidney swelling and blood urine. The majority of stones are composed main ly of calcium oxalate (CaOx). Treatments have been developed to re move kidney stones with minimal renal damage but there is no satisfactory drug to use in clinical therapy. The incidence of kidney stones disease has been increasing over the last years while the age of onset is decreasing. Khella ( Ammi visnaga L., apiaceae) has been used in traditional folk medicine for the treatment and prevention of kidney stones by Egyptia ns as a tea preparati on (24). However, until now there is only one systematic study from Khans group exists which studied a dose of Ammi visnaga .Therefore, the purpose of this dissertation is to investigates the beneficial effects of Khella at a different dose and a single compounds for the prevention of kidney stone formation. We hypothesize that Ammi visnaga L. could inhibit the form ation of kidney stones. In the present study, a tea of KE was prepared by adding 200 mL of boiling water to dried powdered fruits. The mixture was stirred for 5 mi nutes in a covered beak er and then filtered through No.4 filter paper (Whatman International Ltd, Maidstone, England). The extract was lyophilized and kept in -20 C. The identification of KE and quantification of khellin and

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25 visnagin were determined by HPLC-PDA. We wanted to investigate if Khella could inhibit the formation of kidney stone in vitro and in vivo For the in vitro study, we exposed renal epithelial cell lines using MDCK and LLC-PK1 (MardinDarby canine kidney collecting duct tubular epithelial cell line and porcine kidney proximal tubular epithelial cell line, respectively). Cell culture was exposed to Ox (oxalate) and CaOx ( calcium oxalate crystal) with or without the different concentrations of extracts and single compound. The in vivo study was conducted in rats. Ethylene glycol and ammonium chloride we re used to induce kidney stones in the animals for 14 days. The pharmacokinetic study of visnagin, the major compound in KE, was studied in male Sprague-Dawley rats in order to assess the in vivo efficacy and obtain the information about absorption and disposition. The concentration of vi snagin was detected in plasma and urine and pharmacokinetic parameters were calculated. Theref ore, to test the hypoth esis of this study the following specific aims were proposed. Specific aim 1: Identification and quan tification analysis of major compounds in KE. Specific aim 2: Evaluate the preventive effect of KE and single compounds such as khellin and visnagin on oxalate and calcium oxalate crystals exposed to in vitro cell culture. Specific aim 3: Evaluate the prevention of hype roxaluria by an extract of Ammi visnaga L. in stone forming rats. Specific aim 4: Pharmacokinetic analysis of visnagin in rats.

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26 Table 1-1. Salient features of CaOx nephrolithiasis in humans and chronically but mildly hyperoxaluric rats (15-17) Humans Rats Stone composition CaOx monoand dihydrate crystals and organic matrix CaOx monoand dihydrate crystals and organic matrix Matrix composition Carbohydrates, lipids, proteins Carbohydrates, lipids, proteins Matrix proteins OPN* is occluded in crystal; THP** is present between crystals and on their surfaces OPN* is occluded in crystal; THP** is present between crystals and on their surfaces Stone location in the kidneys Renal papillary surface Re nal papillary surface Main cause of nephrolithiasis Hyperoxaluria Hyperoxaluria Inhibitor status Stone former may excrete less citrate and magnesium Urinary excretion of citrate and magnesium is decreased Nucleation Most probably heterogenous Most probably heterogenous Role of genders CaOx stone formers are mostly males Male rats are prone to form CaOx stones Renal injury In idiopathic stone formers, kidney functions are normal but enzymuria may occur Kidney function appears normal; enzymuria occurs concomitantly with hyperoxaluria and nephrolithiasis OPN : Osteoponin, ** THP : Tamm-Horsfall protein

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27 Table 1-2. Effect of Ammi visnga L. (500 mg/kg) in urine output (43) Group and dosage After 5 h D/U D/C After 24 h D/U D/C Control (normal saline: 10mL/kg) 6.15 + 12 45.0 + 0.47 Reference (Urea: 960 mg/kg) 17.60 + 0.74 73.92 + 0.61 Drug ( Ammi visnaga L.: 500mg/kg) 5.30 + 0.41 0.3 0.86 70.1 + 0.43 0.95 1.56 Tabular figures represent the mean + S.E.M. of urine volume (mL/kg) of 6 animals, D/U: Urine output by Ammi visnaga treated rats/Urine output by urea treated rats, D/U: Urine output by Ammi visnaga treated rats /Urine output by control rats. Table 1-3. Effect of Ammi visnaga L. on calcium and oxalate contents of kidney of rats fed with 3% glycol acid for 4 week (43) Treatment Calcium Oxalate Total deposition Protection (test drug 500mg/kg) (mg/100mg of dry kidney weight) (mg/100mg of dry kidney weight) of calcium and oxalate (%) Glycolic acid control 1.69 + 0.10 3.51 + 0.12 5.2 + 0.11 Normal control 0.15 + 0.03** 0.23 + 0.02** 0.38 + 0.01 Ammi visnaga + Glycolic acid 0.72 + 0.13* 0.91 + 0.12* 1.63 + 0.06 74.1 Tabular figures represent the mean + S.E.M. of 6 animals. P < 0.005. ** P <0.0005 (significant relative to glycolic acid control)

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28 Figure 1-1. Structure of the urinary system (4)

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29 Figure 1-2. Normal nephron (7). Structure of the superficial and juxtamedu llary nephrons of the kidney. Nephron represents the function unit of the kidney. PT, proximal tubule; TL, thin limb loop of Henle; MTAL, medullary thinkc ascending limb; CTAL, cortical thick ascending limb; DCT, distal convol uted tubule; CNT, connecting segment; CCD, cortical collecting tubule; OMCDo, coll ecting duct in the outer stripe of outer medulla; OMCDi, collecting duct in inner stripe of outer medulla; IMCD1-3, inner medullary collecting ducts (7).

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30 Figure 1-3. Oxalate action on renal cells (8). [1] Exposure to oxalate elicits a number of membrane changes, including [2] a redistribution of phophatidylserine (PS), altering the membrane surface in such a way to e nhance crystal binding. Attached crystals may then be internalized by endocyt osis. [3] Oxalate-induced membrane perturbations also lead to increased PLA2 activity, which generates two lipid signaling molecules (arachidonic acid and Ly so-PC) and indirectly stimulates the formation of ceramide. [4] Lipid si gnals act on mitichondria dysfunction and increasing production of reactive oxygen species (ROS). [5] Increases in ROS and/or in Lyso-PC trigger changes in gene expre ssion that may serve adaptive functions, [6] which might include proliferation (to repla ce damaged cells) or secretion of urinary proteins (to modulate crystal formation). [7] ROS also promote cell damage which may [8] unmask additional cr ystal binding sites [9] Att ached crystals may form centers for nucleation of new crystals, whic h would favor stone development. Crystal uptake by endocytosis may exacerbate cell damage; alternatively crystals may dissolve within lysosomes or re-emerge at the basolateral surface, again providing centers for stone growth in the renal inte rstitium. [10] Cell de ath produced by oxalate exposure may leave cellular debris that form s a nidus for additional crystals growth, also promoting stone formation [11].

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31 Figure 1-4. Schematic presentation of relationships between various factors, which lead to the formation of idiopathic kidney stone (8, 9).

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32 Figure 1-5. Ammi visnaga L.: Flowering tops, Helsinki Botanical garden, Finland O O OCH3OCH3O CH3 KhellinO O OCH3O CH3 VisnaginOO OCOCH3OCOCH(CH3)CH2CH3CH3CH3 Visnadin Figure 1-6. Main compounds from Khella

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33 CHAPTER 2 IDENTIFICATION AND QUANTIFICAT ION OF COM POUNDS IN KHELL EXTRACT Background Analysis of the plant m aterial is an important step to search of plants with pharmacological activity. The close relative of Ammi visnaga L. (48), Ammi majus L., is often mistaken for Ammi visnaga L. but differs, not only in morphologically but also in its active principles. The major constituents in Khella are khellin and visnagin which do not occur in Ammi majus L. Therefore, khellin and visnagin will be the key compounds in the identification of Ammi visnaga L. Various analytical methods have been used to identify and quantify khellin and visnagin., including thinlayer chromatography (TLC) (49, 50), solidphase extraction (SPF ) (51, 52), capillary electrophoresis (53) and high performance liquid chromatography (HPLC) (54-61). HPLC is the most accepted analytical method for khellin and visnagin. Specific Aim The objectiv e of this study was to identif y and quantify marker compounds which are khellin and visnagin in KE. Materials and Method Chemicals Khellin and Visnagin > 97%, were purchased from Sigma Chemical company (St. Louis, MO, USA). Methanol, ethanol, te trahydrofuran (THF) and ethyl acetate were purchased from Fisher Scientific (Fair Lawn, NJ, USA). All a queous solutions were prepared with deionized water obtained from a NANOPure syst em from Barnstead (Dubuque, IA, USA). Plant Material Dried seeds of Ammi visnaga L. and Ammi Majus L. were collected from Turkey from a commercial provider of plant material in Germ any (Caesar and Laurentz, Hilden, Germany)

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34 Thin Layer Chromatography Analysis 10 cm -length of TLC aluminum sheet were used. 0.5 g each of powered seed of Ammi visnaga L. and Ammi Majus L. were weighed into 40 mL glass beakers to which 10 mL of 60% ethanol was added and stirred by magnetic stirrer bar for 30 mintues. The extract was filtered through No. 4 filter paper (Whatm an International Ltd, Maidstone, England) and was reduced by volume to 5 mL by Rota Evaporator. 20 l of sa mple solution were spotted on TLC plate. For reference materials, khellin and visnagin were dissolved in 1 mL of 60% ethanol and 10 l of each solution were spotted on TLC plate. Ethyl a cetate were used as mobile phase. The spots were revealed by 254 and 365 nm UV light. High Performance Liquid Chromatography with Diode Array De tector (HPLC/DAD) Analysis Sa mples were analyzed using reverse-phase HPLC with diode array detector. ShimadzuHPLC class-vp was used for this work. The instrument was fitted with a diode array detector (SPD-M10 A, VP, Shimadzu). LiChrocart RP -select (250mm x 4 mm i.d., 5m particle diameter) reverse-phase column, with a pr e-column (4.6 mmi.d. x 2.5 cm) was used for a separation of khellin and visnagin. The eluents are (A): water, (B): THF and (C): methanol. The gradient elution program was applied as follo wed: 5-13%B from 0-20 min with constant 5% C balanced with A, 13-22% B and 5-7% C from 20 -52 min balanced with A and then 22-5% B and 7-5% C balanced with A from 52-60 min. The inj ection volume for samples was 10 l. HPLC is carried out at a cons tant temperature 30 C and mobile phase flow ra te of 1 mL/min. Detection wavelength will be set at 330 nm. UV-Vis spect ra were recorded in the range 200-400 nm. Sample preparation for HPLC analysis A 20 g aliqu ot of powdered seed was extracted with 200 mL boiling water in a flask tightly covered with aluminum foil for 5 minutes. The extract was filtered through No. 4 filter paper

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35 (Whatman International Ltd, Maidstone, England) and later freeze dried (FreeZone 6, Labconco, USA). The residue is kept in an amber bottle and stored at -20C. 100 mg of sample was accurately weighed, then transferred to a 5.0 mL volumetric flask and brought to volume with methanol. The sample solution was sonicated fo r 1 min and then filtered through 0.45 m PVDF membrane filter (Milipore Corp.) before analysis. Work solution and the preparatio n of Calibration Standards. Khellin and visnagin (1 mg/mL): The amount of 25.0 mg of khellin and visnagin were accurately weighed, and transferred to a 25.0 mL volumatris flask. The standards were then dissolved in and brought to volume with methanol The standard solutions were filtered through 0.45 m PVDF membrane filter (Mi lipore Corp.) before analysis. Standard solution of khellin, visnagin and three quality controls were prepared in methanol according to Table 2-1. Quantification Calibration was carried out by an external standardization m ethod. Calculation was performed using Microsoft Excel The calibration curves were obtained by plotting the mean area versus the corresponding concentration of the each standard solution. The calibration was considered suitable if not more than 1/3 of the quality control standards showed a deviation from the theoretical values equal or greater than 15%, except at the lower limit of quantification (LLOQ), where it should not exceed 20%. Validation The m ethod was validated over the range of concentration of the ta rget compounds present in the KEs. The validation parameters of linear ity, sensitivity, specificity, precision, accuracy and stability were determined.

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36 The linearity of the calibration curves was de termined by least-squares linear regression method and expressed in term of coefficient of determination (r2). The intra-day precision and accuracy were measured by triplicate analyses of three different concentration levels (low, medium and high) of quality c ontrol standards on the same da y. The precision was based on the calculation of variation (%CV), a nd the accuracy was defined as the percent differences between the theoretical and measures valued. The limit of quantification for the assay was defined as the minimum concentration of quality controls. Results Thin Layer Chromatograohy Analysis Khellin and visnagin we re showed on the TLC plate from the spot of Ammi visnaga L.. However, they were not in the spot of Ammi majus L.(Figure 2-1). High Performance Liquid Chromatography Analysis Identification Khellin and visnagin cou ld be detect ed in the HPLC chromatogram of Ammi visnaga L. but not in the HPLC Chromatogram of Ammi majus L. (Figure 2-2). Linearity Calibration curves (n = 9) operating in th e range of 0.05-1.0 mg/mL for both Khella components were linear (r2 > 0.99) (Figure 2-3). Sensitivity In this study, the limit of quantification (LLOQ) is defined as the lowest concentration for quality control. The LLOQ of khellin and visnagin were 0.01 mg/mL. Specificity The method provided good reso lutions between khellin a nd visnagin. Peaks of both compounds had similar retention times and UV spectra (200-400 nm) when compared to the

PAGE 37

37 standards (Figure 2-2). There was no endogenous interference from KE (Figure 2-3) in this assay, indicating specificity of the methods to the tested compounds. Additionally, the UV spectra of all tested compounds showed more than 98% of similarity with those obtained using the respective standard compounds. Precision and Accuracy The precision intraand inter-day for khellin and visnagin were sa tisfactory with CV values between 0.93 and 12.98%. Similarly, the accu racy of the assay was between 93.45 and 105.78% for all compounds tested at three different concentrations. The results are summarized in Table 2-2. Stability The standard solutions of khellin and visnagin were found stable on autosampler at 20 C (Table 2-3). Quantitation of Khellin and Visnagin in KE The results from HPLC/DAD showed that the amount of khellin and visnagin are 28.1 0.4 and 17.2 0.1 mg/g freeze dried extract, respectively Table 2-4. Discussion and Conclusion This study reported an identification of Ammi visnaga L. by using the TL C-plate and HPLC/DAD. The comparison of each compound was performed by a comparison with available standards and UV evaluation. This approach made it possible to distinguish between Ammi visnaga L. and Ammi majus L.. The HPLC profiles of the extracts are shown in Figure 2-2 A with a profile of khellin and vi snagin at 330 nm. The quantita tive HPLC/DAD findings of khellin and visnagin are 28.1 0.4 and 17.2 0.1 mg/g freeze dried extract, respectively. The developed method is appropriate to complete characteriza tion and quantification of marker compounds in

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38 KE. The drug extract ratio is 5:1 calculated by pr epared tea 50g from Kh ella seed and got 10 g of extract.

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39 Table 2-1. Concentrations of the standard solu tions used for the calibration curves and quality controls (QCs) of khellin and visnagin Compounds Standard Solution (mg/mL) QC (mg/mL) Khellin 0.05, 0.1, 2.5, 0.5, 0.75, 1.0 0.075, 0.4, 0.8 Visnagin 0.05, 0.1, 2.5, 0.5, 0.75, 1.0 0.075, 0.4, 0.8 Table 2-2. Intra-day (n=3) and inter-day (n=9) assay parameters of khellin and visnagin. Precision expressed as %CV and accuracy expressed as % of the theoretical concentration. Khellin QC1-0.075 mg/mL QC1-0.4 mg/mL QC1-0.8 mg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Intra-day precision 5.93 4.68 10.32 6.57 2.13 7.18 2.62 0.93 3.76 Accuracy 101.51 105.8 95.57 102.94 100.45 105.34 98.76 105.78 101.21 Inter-day precision 13.75 9.67 8.73 Accuracy 98.99 100.52 105.73 Visnagin QC1-0.075 mg/mL QC1-0.4 mg/mL QC1-0.8 mg/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Intra-day precision 3.88 7.97 12.98 8.44 3.66 8.53 5.45 4.75 10.79 Accuracy 102.95 104.3 97.88 93.45 97.84 102.56 96.87 103.21 99.78 Inter-day precision 10.12 2.56 9.89 Accuracy 95.67 100.67 97.63

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40 Table 2-3. Stability test of khellin and vi snagin after 24 hours on autosampler at 20 C. Data represents the per centage remaining of all test compounds. Compounds % Remaining on autosampler at 20C within 24 hour QC10.075 mg/mL QC20.4 mg/mL QC30.8 mg/mL Khellin 91.71 + 0.92 100.57 + 2.12 98.58 + 0.67 Visnagin 92.57 + 3.93 101.12 + 5.74 94.59 + 3.56 Table 2-4. Quantitative analysis of khellin and vina gin in KE (water extract). Data represents the mean SD mg/g freeze-dried extract. Khellin Visnagin mg/g Average SD mg/g Average SD 27.51 17.16 28.13 17.32 28.51 28.1 0.4 17.24 17.2 0.1

PAGE 41

41 Rf = 0.78 Rf = 0.74 Rf = 0.75 Rf = 0.79 Rf = 0.95 Rf = 0.79 Rf = 0.75 Rf = 0.74 Rf = 0.78 Rf = 0.95K VAV AM Figure 2-1. Representation of Thin La yer Chromatography plate result of Ammi visnaga L. and Ammi majus L.

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42 Minutes 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 mAu 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 SPD-M10Avp-330 nm water extract nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 mAu -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 mAu -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 29.96 Min water extract nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 mAu-100 0 100 200 300 400 500 600 700 800 900 mAu-100 0 100 200 300 400 500 600 700 800 900 33.20 Min water extract Vis nag in Kh e llin Minutes 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 mAu 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 SPD-M10Avp-330 nm water extract nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 mAu -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 mAu -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 29.96 Min water extract nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 mAu-100 0 100 200 300 400 500 600 700 800 900 mAu-100 0 100 200 300 400 500 600 700 800 900 33.20 Min water extract Minutes 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 mAu 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 SPD-M10Avp-330 nm water extract nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 mAu -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 mAu -100 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 29.96 Min water extract nm 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 mAu-100 0 100 200 300 400 500 600 700 800 900 mAu-100 0 100 200 300 400 500 600 700 800 900 33.20 Min water extract Vis nag in Kh e llin Minutes 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 mAu0 100 200 300 400 500 600 700 800 900 1000 SPD-M10Avp-330 nm Ammi majus nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 200 400 600 800 1000 1200 1400 mA u 0 200 400 600 800 1000 1200 1400 31.91 Min Ammi majus nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 250 500 750 1000 1250 1500 mAu 0 250 500 750 1000 1250 1500 25.78 Min Ammi majus Minutes 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 mAu0 100 200 300 400 500 600 700 800 900 1000 SPD-M10Avp-330 nm Ammi majus nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 200 400 600 800 1000 1200 1400 mA u 0 200 400 600 800 1000 1200 1400 31.91 Min Ammi majus Minutes 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0 52.5 55.0 57.5 60.0 mAu0 100 200 300 400 500 600 700 800 900 1000 SPD-M10Avp-330 nm Ammi majus nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 200 400 600 800 1000 1200 1400 mA u 0 200 400 600 800 1000 1200 1400 31.91 Min Ammi majus nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 250 500 750 1000 1250 1500 mAu 0 250 500 750 1000 1250 1500 25.78 Min Ammi majus (A) Ammivisnaga L. (B) Ammimajus L. Figure 2-2. An HPLC chromagram. A) Ammi visnaga L. B) Ammi majus L.

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43 A B Figure 2-3. Mean Calibration curves of compounds in Khella extract (n=9). A) Khellin and B) Visnagin. Vertical bars represent the standard deviations (SD) of the means y = 1E+07x 9143.9 R2 = 0.9912 0 2000000 4000000 6000000 8000000 10000000 12000000 00.20.40.60.811.2 Khellin (mg/ml)Area y = 1E+07x 14625 R2 = 0.9911 0 2000000 4000000 6000000 8000000 10000000 12000000 14000000 00.20.40.60.811.2 Khellin (mg/ml)Area

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44 nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 250 500 750 1000 1250 1500 mAu 0 250 500 750 1000 1250 1500 29.99 Min water extract khellin_STD.spc 29.69 Min Calibration4-0.5mg/ml nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 200 400 600 800 1000 mAu 0 200 400 600 800 1000 33.40 Min water extract visnagin_STD.spc 33.22 Min Calibration4-0.5mg/ml A. Similarity: 0.9997 B. Similarity: 0.9973 1 2 2 1 Figure 2-4. Absorbance-wavelength spectras. A) Khellin. B) Visnag in. (1) represents the spectra of the peak with the same retention time of the corresponding standard but obtained after injection of KE and (2) represents the spectra of the standard compound.

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45 CHAPTER 3 EVALUATE THE PREVENTIVE EFFECT OF KEHL LA EXTRACTS ON OXALATE AND CALCIUM OXALATE CRYSTALS EXPOSED TO IN VITRO CELL CULTURE Background The use of renal epithelial cells in tissue cultur e is widely accepted as a powerful tool in physiological and cell biological studies. A num ber of permanently growing cell lines with characteristics of distinct nephronal segments ar e presently available (62). Over the past few decades two continuous renal epithelial cell lines have been most used or studying nephrolothiasis, the Mardin-Darby canine kidney collecting duct tubular epithelial cells (MDCK) and porcine kidney proxim al tubular epithelial ce lls (LLC-PK1). MDCK cells have been widely used as a model system for th e distal/collecting duct and LLC -PK1 cells have retained many characteristics of the proximal tubule (62-67). Lactate dehydrogenase, also ca lled lactic dehydrogenase, or L DH, is an enzyme found in the cells of many body tissues, including the heart, liver, kidneys, skeletal muscle, brain, red blood cells, and lungs. It is responsible for conver ting muscle lactic acid into pyruvic acid, an essential step in producing cellu lar energy. When disease or injury affects tissues containing LDH, the cells release LDH, where it is identified in higher than normal levels. Therefore, in this study, LDH was used as a biomarker to detect cell damage (68). Specific Aim The objectiv e of this study was to evaluate the preventive effect of KE in in vitro cell culture using MDCK and LLC-PK1 cell lines. Materials and Methods Cell lines, Chemicals and Biochemicals MDCK (Madin-Canine Kidney co llecting duct tubular epithelium cell line) and LLC-PK1 (Porcine Kidney Proximal Tubular epithelial ce ll line) cells were provided by Dr. S. Khan

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46 (Department of Pathology, College of Medicine). Kh ellin (97%), visnagin (97%), oxalic acid and calcium oxalate monohydrate were purchased fr om Sigma Aldrich. Dulbeccos modified eagless medium/F-12, calf serum, antib iotics-antimycotic, 0.05% Trypsin, 0.25% ethylenediaminentetraacetic aci d, insulin/transferin/selenium mix, hydrocortisone, 3.4 mL triiodo-L-thyronine and prostagla ndin E1were purchased from Fisher Scientific. A CytoTox 96 Non-Radioactive Cytotoxicity a ssay kit was purchased from Fisher Scientific. The assay was performed according to manuf acturers instructio n. All other reagents are from Sigma. Plant Material Dried f ruit of Ammi visnaga L. were collected form Morocco from a licensed commercial provider of plant material in Germany (Caesar and Laurentz, Hilden, Germany) Extraction procedure An extract o f Ammi visnaga L. was prepared in the procedure as in chapter 3. Sample preparation The extract was suspend ed in acclimizati on medium (500 mL Dulbeccos modified eagless medium/F-12, 10 mL antibiotics-antimyco tic solution, 5 mL insulin/transferin/selenium mix, 1mL hydrocortisone, 3.4 mL triiodo-L-thyr onine and 1mL prostaglandin E1) in the concentrations of 125, 250 and 500 mg/mL. Cell Culture Experiment MDCK (Madin-Canine Kidney co llecting duct tubular epithelium cell line) and LLC-PK1 (Porcine Kidney Proximal Tubular epithelial cell line) cel ls were used in this experiment. Cell cultures were exposed to oxalate (Ox) at concen tration of 300 mol and calcium oxalate (CaOx) crystals at concentration of 133 g/cm2. These concentrations were based on the previous data study. LLC-PK1 and MDCK were maintained in growth medium (500 mL Dulbeccos modified eagless medium/F-12, 50 mL newb orn calf serum and 10 mL antib iotics-antimycotic) in 75 cm2

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47 T-Flask (Corning, Corning, New York). The cells were subcultured by disassociation with 0.05% Trypsin and 0.25% ethylenediaminentetraacetic acid and seeded to 24 well-plate tissue culture dishes. Dose response experiment of Khella After achiev ing confluence the cells were wean ed from growth medium to acclimization medium. Cells were divided to 11 groups (n=6), pretreatment for 24 hours and treatment as Table 3-1. The cells were exposed for 1, 3, 6, an d 12 hours with or without KE then the spent media from individual wells were recovered a nd centrifuged to remove crystal and cellular debris. The media were analyzed for lactate dehydrogynase (LDH) enzyme to determine cell viability through membrane damage. Pure compound experiment After obtaining the appropriate concentration of KE from the dose response experiment of KE, khellin and visnagin were quantitively calcu lated based on HPLC analysis. Then, LLC-PK1 and MDCK were exposed to khellin or visnag in in the same method as the dose response experiment of KE. The details of the dose regimen are listed in Table 3-2. Lactate dehydrogenase (LDH) Assay Medium were aliquoted to designated wells of 96-well plate. A CytoTox 96 NonRadioactive Cytotoxicity assay ki ts (Promega, Fisher Scientific) was used to determine % LDH release. Substrates supplied with the kit were added to all samples (in duplicate), positive control (MDCK or LLC-PK1 cells were lysed with lysi s solution supplied with the kit) and blank (acclimization medium). The plate was incubated at room temperature for 30 minutes in the dark. Stop solution supplied with the kit was added to all samples, positive control and blanks. Optical density absorbency was read at 490 nm on a micr oplate reader (Synergy HT, Biotech instrument INC., VT).

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48 Statistical analysis Statis tical calculations were carried out w ith one-way ANOVA with the Newman-Keuls test for multiple comparisons (GraphPad Prism 5). The results were considered significant if the probability of error was < 0.05. Results Dose Response Experiment of Khella LLC-PK1 As can be seen from Figure 3-1, the upper panel showed % LDH release from oxalate exposure to LLC-PK1 with or without KE. At every time point, the result showed that % LDH release after trea tment with 100 and 200 g/mL KE signi ficantly decreased comparing to Ox group. While the below panel showed % LDH releas e from CaOx crystals exposure to LLC-PK1 with or without KE, % LDH release after treatment w ith 50, 100 and 200 g/mL KE significantly decreased compared to the CaOx crystals group. MDCK It is shown in Figure 3-2 that 100 and 200 g /mL KE can reduce % LDH release from the cell when exposed to oxalate as well. There were significant decreases of the % LDH after treatment for 1 and 3 hours; whereas, % LDH re lease was reduced significantly after treatment with 100 g/mL KE at every time point. Only at the 1 hour treatment with 200 g/mL KE was there a significant decrease with CaOx crystals exposure to MDCK. KE potentially prevents LLC-PK1 and MDCK cells from oxalate or calcium oxalate crystals induced cell in jury due to the decreased amount of % LDH release. Comparing % LDH released from treated cells between 100 and 200 g/mL KEs was not significantly different. Therefore, we chose 100 g/mL KE as the approp riate concentration to calculate the amount of khellin and visnagin for exposure to LLC-PK1 and MDCK.

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49 Pure Compound Experiment LLC-PK1 OX or crystals exposure to LLC-PK1 with khellin and visngain, % LDH released was decreased at 1, 3 and 6 hours com pared to OX or CaOx crystal groups. Therefore, khellin and visnagin may reduce % LDH releas ed from LLC-PK1 (Figure 3-3). MDCK Ox exposure to MDCK with khellin or vi snagin did not decrease % LDH released compared to Ox or CaOx crystal groups. While with CaOx crystals esposure to MDCK with or without khellin and visnagin, there was only sign ificant decreased % LDH released for visnagin at 1 and 6 hours (Figure 3-4). Discussion and Conclusion The interaction between renal ep ith elial cells and oxalate or calcium oxalate crystals plays a significant role in the formation, retention a nd development of calcium oxalate stone disease (69). In the present study, oxalate or calcium oxalate crystals e xposure resulted in a significant increase of LDH release (an indicator of cell injury). The cellular injury potentiates calcium oxalate crystal formation (69). The experiment s were performed using Ox or CaOx monohydrate crystals, since CaOx crystals in the intact orga n can not exist in the ab sence of Ox. Our data showed that LLC-PK1 and MDCK cells were inju red when exposed to Ox or CaOx crystals which correlates with results from other studie s (70-75). Studies from ot her laboratories have likewise indicated that Ox and CaOx crystals are injurious to renal epithel ial cells in culture (62, 75, 76). 100 and 200 g/mL of KE extract reduced LDH re lease from LLC-PK1 exposure to Ox or CaOx crystals at every incubation time a nd also reduced LDH release from MDCK exposure to Ox or CaOx at 1 and 3 hour. It is suggested that KE protec ts against Ox or CaOx crystal induced cell damage to renal epithelial cell a nd is a greater effect on LLC-PK1 (represents

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50 proximal tubular epithelial cells) than MDCK (represents collec ting tubule epithelial cell). The active compounds, khellin and visnagin were inve stigated in this study as well. They were calculated based on the quantific ation from chapter 2 and 100 g/mL KE. Khellin and visnagin were calculated based on 100 g/mL of KE because it is not significant difference in LDH release from 200 g/mL KE. Therefore, 2.81 and 1.72 g of khellin and vi snagin, respectively, were used. The results revealed that khellin and visnagin decreased LDH released from both LLC-PK1 and MDCK when exposed to Ox or Ca Ox crystals. The results showed that the protective effect is more effec tive in LLC-PK1 than MDCK cells but not significant difference to KE (Figure. 3-3). The mechanism of action of KE for preventi on of cell damage has not been elucidated. Khan et al investigated the diuretic activity of Ammi visnaga L. in rats and found that the volume of urine output from the group of animals treated with 500 mg/kg Ammi visnaga L. was almost the same from the reference group treated with urea. It means that KE could have diuretic activity to remove stones from the urological system. In addition, Khan et al. (43) studied the deposition of calcium and oxalate in kidney and found that KE could redu ce the renal content of calcium and oxalate. Therefore, diuretic activity might be one of the possible mechanisms of action of KE. In addition, khellin and visnagin have spasmolyti c activity that could relax the smooth muscle which might extend the urological tube and help remove stones easily from the urological system (42, 77). Khellin and visnagin are coumarins. From their structures in Figure 1-6., calcium ion might attach at the methoxy group of khellin and visnagin. The new complex might be removed from the urological system or prevent the fo rmation of calcium oxalate. Moreover, another compound in KE is visnadin which is a cor onary vasodilator and a positive inotropic,

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51 bradycardic and spasmolytic agent; this is proba bly a result of its calcium blocking activity, shown in vitro (33). KE has also been used to ease th e pain of kidney stones as they scrape through the ureter the small t ube that leads from the kidney to the urinary bladder (43). In conclusion, our data indicated that an aque ous extract of Khella, as well as khellin and visnagin prevent cell damage caused by Ox or CaOx crystals and is more effective to LLC-PK1 than MDCK cells. However, the exact mechanis m of action is still unknown. Khellin, visnagin and visnadin could be the key co mpounds in preventing cell injury.

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52 Table 3-1. Dose regimen for dose respons e experiment of KE in cell culture Group Pretreatment Treatment 1 AM AM 2 AM AM + 300 M Ox 3 AM + 5 g/mL KE AM + 300 M Ox + 5 g/mL KE 4 AM + 10 g/mL KE AM + 300 M Ox + 10 g/mL KE 5 AM + 100 g/mL KE AM + 300 M Ox + 100 g/mL KE 6 AM + 200 g/mL KE AM + 300 M Ox + 200 g/mL KE 7 AM AM+ 133 g/cm2 COM 8 AM+ 5 g/mL KE AM + 133 g/cm2 COM + 5 g/mL KE 9 AM + 10 g/mL KE AM+ 133 g/cm2 COM + 10 g/mL KE 10 AM + 100 g/mL KE AM + 133 g/cm2 COM + 100 g/mL KE 11 AM + 100 g/mL KE AM + 133 g/cm2 COM + 100 g/mL KE AM=Acclimization Medium. Ox=Oxalate. COM=Calcium oxalate monohydrate.

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53 Table 3-2. Dose regimen for pure compound experiment Group Pretreatment Treatment 1 AM AM 2 AM AM + 300 M Ox 3 AM + KE AM + 300 M Ox + 100 g KE 4 AM + Khellin AM + 300 M Ox + 2.81 g Khellin 5 AM + Visnagin AM + 300 M Ox + 1.72 g Visnagin AM=Acclimization Medium. Ox=Oxalate COM=Calcium oxalate monohydrate.

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54 Figure 3-1. The % LDH release from oxalate or calcium oxalate crystals exposure to LLC-PK1 with or without KE (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001). 1 h 0 2 4 6 8 10* *% LDH release 3 h 0 2 4 6 8 10*** **%LDH Release 6 h 0 2 4 6 8 10Control 300 M Ox 300 M Ox + 10 g/mL KE 300 M Ox + 50 g/mL KE 300 M Ox + 100 g/mL KE 300 M Ox + 200 g/mL KE *** ***%LDH Release 0 3 6 9 12 15** **%LDH release 0 3 6 9 12 15** **%LDH Release 0 3 6 9 12 15Control 133 ug/cm2 COM 133 ug/cm2 COM+ 10 g/mL KE 133 ug/cm2 COM + 50 g/mL KE 133 ug/cm2 COM+ 100 g/mL KE 133 ug/cm2 COM+ 200 g/mL KE *** *** ***% LDH Release

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55 Figure 3-2. The % LDH release from oxalate or calcium oxalate crystals e xposure to MDCK with or without KE (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001). 1 h 0 10 20 30 40** *%LDH release 3 h 0 10 20 30 40*** *%LDH Release 6 h 0 10 20 30 40Control 300 M Ox 300 M Ox + 10 g/mL KE 300 M Ox + 50 g/mL KE 300 M Ox + 100 g/mL KE 300 M Ox + 200 g/mL KE %LDH Release 0 3 5 8 10** ***%LDH release 0 3 5 8 10***%LDH release 0.0 2.5 5.0 7.5 10.0 12.5 15.0Control 133 ug/cm2 COM 133 ug/cm2 COM + 10 g/mL KE 133 ug/cm2 COM + 50 g/mL KE 133 ug/cm2 COM + 100 g/mL KE 133 ug/cm2 COM + 200 g/mL KE *%LDH release

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56 Figure 3-3. The % LDH release from oxalate or calcium oxalate crystals exposure to LLC-PK1 with or without pure compounds (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001). 1 h 0 5 10 15 20** ** *%LDH release 3 h 0 5 10 15 20*** *** **%LDH release 6 h 0 5 10 15 20*** *** ***Control 300 M Ox 300 M Ox + 100 g/mL KE 300 M Ox + 2.81 g/mL Khellin 300 M Ox + 1.72 g/mLVisnagin %LDH release 0 5 10 15 20* ******%LDHrelease 0 5 10 15 20*** *** ***%LDH release 0 5 10 15 20*** ** ***133 cm2 COM+ 2.81 g/mL Khellin Control 133 cm2COM 133 cm2 COM+ 100 g KE 133 cm2COM+1.72 g/mL Visnagin %LDH release

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57 Figure 3-4. The % LDH release from oxalate or calcium oxalate crystals exposure to MDCK w ith or without pure compounds (comparing to Ox or CaOx group, p < 0.5, ** p< 0.01, *** p< 0.001). 0 5 10 15 20 25*** *%LDH release 0 5 10 15 20 25*%LDH release 0 5 10 15 20 25*** ***133 cm2 COM+2.81 g/mL Khellin Control 133 cm2 COM 133 cm2 COM + 100 g/mL KE 133 cm2COM +1.72 g/mL Visnagin %LDH release 1 h 0 5 10 15 20 25*%LDH release 3 h 0 5 10 15 20 25*%LDH release 6 h 0 5 10 15 20 25Control 300 M Ox 300 M Ox + 100 g/mL KE 300 M Ox + 2.81 g/mL Khellin 300 M Ox+ 1.72 g/mL Visnagin %LDH release

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58 CHAPTER 4 EVALUATE THE PREVENTION OF HYPE R OXAURIA BY AN EXTRACT OF Ammi visnaga L. AND MAJOR COMPOUNDS IN STONE FORMING RATS Background Hyperoxaluria, defined as exces sive urinary oxalate, is the m ain risk factor for human idiopathic CaOx stone formation and induction of hyperoxaluria is essential for the development of CaOx urolithiasis in rat (78). Calcium oxala te stone is the most prevalent component in urolithiasis and many experimental models have been used to demonstrate its formation in the animal kidney (79, 80). Ethylene gl ycol (EG), which is a precur sor to oxalate formation, has generally been used in combina tion with ammonium chloride (NH4Cl) or vitamin D3 in an attempt to form calcium oxalate crystals in ur ine and calcium oxalate de posits on the kidney of rats (14). Urinary excretion of oxalate increased during the ch ronic administration of ethylene glycol (EG) as a 0.75% aqueous solution in drinking water to male Sprague-Dawley rats. Excretion of calcium, magnesium, and ci trate decreased concomitantly (17). Ammi visnaga L. or Khella has a long tradition as use for treatment of kidney stone disease (29, 31). However, few scientific investigations ha ve been performed in the field of kidney stone disease. Khan et al studied a 500mg/kg dose of Ammi visnaga L. animals and found that it showed the diuretic effect and reduced the calcium oxalate conten ts in kidney (43). The present study was performed with 3 doses of KE and also determined of calcium, citrate and oxalate, major chemistry in urine for kidney stone. In a ddition, two active compounds of KE, khellin and visnagin, were investigated in stone forming rats. Specific Aim The objectiv e of this study was to evaluate the preventive effect of KE and its active compounds in experimentally i nduced nephrolithiasis rats.

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59 Materials and Methods Chemicals and Biochemicals Khellin, visnagin (97%), ethylene glycol (E G), potassium citrate monohydrate (PCi), and carboxymethylcellulose (CMC) were purchase from Si gma Aldrich (St. Louis, MO, USA). Citric acid determination kit was purch ase from R-biopharm, Oxalate ki t was purchased from Trinity biotech. Calcium determination kit was purchased from Bioassay system (CA, USA). Lactate dehydrogenase, alkaline phosphatase reagent kits were purchased from Vitalab and used to perform the analysis on VitalS electra 2 chemistry analyzer according to manufacturers instruction. Plant Material and Extraction Procedure An extract o f Ammi visnaga L. was prepared in the procedure as in chapter 2. Sample preparation The extracts were suspended in 0.8% carboxy m e thylcellulose (Sigma Aldrich) and given orally in the dose of 125, 250 and 500 mg/kg once da ily. Khellin and visnagin in the dose of 5 and 10 mg/kg were suspended in 0.8% CMC and gi ven orally to the anim al once daily. Ethylene glycol and ammonium chloride were added in drinking water to make a concentration of 0.75% and 1.0% respectively. Potassium citrate was used as the positive control ( 81-85) and prepared at the concentration of 2.5 g/kg in 0.8% CMC. Animals and Experimental Protocols Male Sprague-Dawley rats, weighing 150-200 g were purchased from Harlan (IN, USA) and divided into the experimental groups (8 ra ts per group) according to table 1. The animals were housed in plastic cages and were allowed to adapt to their environment for one week before used for experiments. All the animals were ma intained on 12hr/12hr light/dark cycle. They received a standard chow and water ad libitum during experimentation. All animal experiments

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60 were performed according to the policies and guide lines of the Institutional Animal Care and Use Committee (IACUC) of the University of Flor ida, Gainesville, USA (NIH publication #85-23). The animals were given the treatment in tabl e 4-1 orally once daily for 14 days with KE. Khellin and visnagin were also given to the other group of animal as Table 4-2 describes. The animals were placed in the metabolic cage for urine collection at day 0, 7 and 14. Oxalate, citrate, calcium, lactate dehydroge nase (LDH) and alkaline phospha tase (ALP) were tested in urine by the commercial kit according to the manufacturers instructions. The urine was centrifuged at 2800 rpm for 10 minute and the su pernatant was transfe rred for testing the chemistry at the same day. After 14 days, the an imals were sacrificed and the kidneys were harvested for histopathological assay of calcium oxalate depositi on. The kidneys of animals that died before 14 days were also harvested and determined the calcium oxalate depositions as well. Oxalate, citric acid a nd calciu m determination Trinity Biotech (New Jersey, USA) Oxalate r eagents were used for the quantitation of oxalate in urine. R-Biopharm citric acid determination kit (Michigan, USA) was used for quantitation of urinary citrate. Quantichrom Calc ium determination (CA, USA) kit was used for quantitation of calcium. Lactate dehydrogynase and alkaline phosphatase assay Lactate dehydrogenase (LDH) a nd alkaline phosphatase (ALP) were tested on the Vitalab Selectra 2, chem ical analyzer. Calcium oxalate crystal deposition in kidney After harvesting, the kidney was kept in form alin, sliced vertically and sent to histology services (Gainesville, Fl) for H&E staining. Calc ium oxalate crystal depos itions were count by microscopy and given a score as follows; <1= 0, <10 = 1, <30 = 2, <50 = 3, < 75 = 4 and 75 = 5.

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61 Statistical analysis Data were analyzed by one-way ANOVA and the Newm an-Keuls test for multiple comparisons using GraphPad Prism 5.0 software, San Diego, USA. The results were considered significant if the probabi lity of error was < 0.05. Results Khella Experiment At day 0, all of the chemistry results which are L DH, ALP, calcium, oxalate, citric acid,pH and urine from every treatment groups were not significant different comp aring to the control group. Survival rate The first anim al of EG group di ed on day 8 and 2 animals were left until the end of the study (Figure 4-1). While all of the animals from the other groups were st ill alive until the last day of the experiment, except an animal from 500 mg/kg KE died at day 8 which was not statistically significant. Regarding these results KE protects animals from kidney stone induced by EG. However, the animals in the EG group at day 14 were not sufficient to compare the statistics; we compared the urine chemistry assays at day 7. Lactate dehydrogenase and alkaline phosphatase Lactate dehydrogenase and alka line phosphatase in urine were not of significant difference in all treatm ent groups (F igure 4-2 and Figure 4-3). Urine volume Calcium, oxalate, citrate, and pH There were no significant diffe rences in the urine volum e and urinary calcium content from all animals (Figure 4-4, 4-5). The urin ary excretion of oxalate (Figure 4-6) was significantly decreased along with a significant increase of urin ary citrate (Figure 4-7). In

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62 addition, the urinary pH was increased in a dose-de pendent manner which is correlated to urinary citrate excretion (Figure 4-8). Calcium oxalate crystal depositions There was no incidence of calcium oxalate cr ystal deposition in the control group; whereas there was a high incidence of calcium oxalate crystal deposition in the EG group (Figure 4-9). The animals that additionally received potassium citrate and all dose of KE significantly scored lower for calcium oxalate crystal deposition th an the EG group. The histopathological kidneys were shown in Figure 4-10. Major Compound Experiment The sam ples from in vivo experiment of khellin and visnagin were analyzed for urinary oxalate, citrate, pH and calcium oxalate crystal deposition in kidne ys. All of the rats from this experiment survived. There were no significant differences of any da ta from urine sample at day 0. There were no significant di fferences in urinary calcium and oxalate in every group of treatment at day 7 and 14 (Figure 4-11 and 4-12) Urinary citrate signifi cantly increased for animals treated with 2.5 g/kg potassium citrate at day 7 but there was nothing significant of urinary citrate at day 14 (Figur e 4-13). Urinary pH was only si gnificantly increased in the 2.5 g/kg potassium citrate group at day 7 and day 14 (Figure4-14). However, calcium oxalate crystal deposition scores from the animals that receive d 2.5g/kg potassium citrate, 5, 10 mg/kg khellin and 5, 10 mg/kg of visnagin were significant lowe r than the animal that received only 0.75% EG and 1.0% NH4Cl (Figure 4-15). Discussion and Conclusion The results show that KE could prevent nephr olithiasis in stone for ming rats by lowering in the urinary excretion of the oxalate along with an increase of ur inary excretion of citrate. In addition, the urine pH was increased in a dose-de pendent manner which correlates to an increase

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63 of urinary citrate; therefore, the calcium oxalate crystals in animals receiving KE could not be formed in the kidney. Khan et al repo rted that the prophylactic activity of Ammi visnaga to kidney stone disease might be attributed to the di uretic activity (43). However, our result did not show the diuretic acidity. The mechanism of act ion has not yet been elucidated. Although khellin and visnagin did not increase th e urinary citrate and pH, the cal cium oxalate crystal deposition scores were signifi cantly decreased. Potassium citrate (PCi, 2.5g/kg) was used as a positive control (84, 86, 87). The results showed that PCi increased urinary citrate along with an increase of urinary pH and oxalate. It is indicated that PCi has been form ed with calcium and excreted as calcium citrate; therefore, leading to calcium oxalate can not be formed and oxalate can be excreted in the urine (88, 89). In the present study, KE increased urinary citrat e in a dose dependent manner. 500 mg/kg KE resulted in a higher urinary citrate compared to PCi, however the CaOx deposition score of KE treatment is not different from PCi treatment Interestingly, the CaOx deposition score from khellin and visnagin treatment are not significant di fferent to PCi treatment but the urinary citrate from both treatments are lower than PCi treatm ent. Therefore, KE, khellin, visnagin and PCI might have a different mechanism of action to prevent the formation of calcium oxalate stone. Interestingly, the final stage comes up with th e same effective to prevent the kidney stone. Urinary citrate plays an important role in reducing recurrences of calcium oxalate stone. Its inhibitory effect on calcium oxalate crystalli zation is generally recognized, the mechanism of action being a reduction of calcium oxalate supers aturation by formation of complexes with calcium and direct inhibition of crystal growth with aggregation and increasing urinary pH (81, 84, 87). Our results are relative to these reports that lead to inhibition of calcium oxalate crystal deposition in rat kidneys. There are several fact ors that increase urin ary citrate including,

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64 decrease of dietary protein intake, administration base, such as potassium citrate and increase of hormone modulation turnover, such as estrogen lead ing to an increase of citrate excretion (81, 83-87, 90-93). In addition, systemic acid-base status is a dominant key to ci trate excretion. It has long been known that acidosis de creases citrate excretion, whereas alkalosis incr eases it. Most filtered citrate is taken up across the apical membrane of the proximal tubule via a sodiumdicarboxylate co-transport (NaD C-1), Figure 4-16 (81, 82, 85, 94-96) It also enters proximal tubular cells across the basolatera l membrane. This NaDC-1 carries 3 Na ions with each citrate ion. The preferred substrate for NaDC-1 is citrate2+, whose abundance increas es along the length of the proximal tubule as the luminal fluid become s more acidic, a decrease in citrate excretion occurs. The proximal tubule reabsorbs approximate ly 75% of filtered citr ate and thus is the primary determinant of final urin ary citrate excretion (81). Change in systemic pH would alter intracellular pH, resulting in cha nges in intracellular citrate metabolism, leading to alterations in citrate reabsorption and hence urinar y citrate excretion (Figure 4-17). Our data demonstrated that KE increased the urine pH in a dose dependent manner along with an increase of urinary citrate. It could be possible that KE changed the luminal pH and thus the reabsorption of citrate was i nhibited leading to the prevention of CaOx crystal formation in kidney. However, the active compound experiment results showed that ur inary citrate and pH were not increased from khellin and visnagin treatment; the calcium oxalate crystal deposition scores were decreased. Thus, the inhibition of calcium oxalate stone formation of khellin and visnagin might not interfere with the citrate r eabsorption. It might be a result of its calcium blocking activity, shown in vitro (37). This can inhibit vascul ar smooth muscle contraction, dilate peripheral and coronary vessels and increase coronary ci rculation (33, 36, 38-40). Visnagin also has negative chronotropic and inot ropic effects and reduces peripheral vascular

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65 resistance (33, 41). The khellin also acts as a vasodilator and ha s bronchodilatory and spasmolytic activity (36, 38, 42). Based on these resu lts it would be possible that there are other actives compounds in the extract interferes th e citrate metabolism. In conclusion, the in vivo experiment indicated that KE and its compounds, khellin and visnagin, has potential for use in preventing form ation of calcium oxalate nephrolithiasis but it is likely that different mechan ism of action are involved.

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66 Table 4-1. Dose regimen of KE Group Treatment N 1 Control 8 2 EG + NH4Cl 8 3 EG + NH4Cl + 2.5 g Potassium citrate 8 4 EG + NH4Cl + 125 mg/kg KE 8 5 EG + NH4Cl + 250 mg/kg KE 8 6 EG + NH4Cl + 500 mg/kg KE 8 EG= .75% Ethylene glycol in drinking water NH4Cl=1.0% Ammonium Chloride in drinking water Table 4-2. Dose regimen of khellin and visnagin Group Treatment N 1 Control 4 2 EG + NH4Cl 6 3 EG + NH4Cl + 2.5 g Potassium citrate 6 4 EG + NH4Cl + 5 mg/kg Khellin 8 5 EG + NH4Cl + 10 mg/kg Khellin 8 6 EG + NH4Cl + 5 mg/kg Visnagin 8 7 EG + NH4Cl + 10 mg/kg Visnagin 8 EG= .75% Ethylene glycol in drinking water NH4Cl=1.0% Ammonium Chloride in drinking water

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67 Survival 0 2 4 6 8 10 12 14 0 50 100 150 Control 0.75% EG+ 1%NH4Cl 2.5 g/Kg Potassium Citrate 125 mg/Kg Khella extract 250 mg/Kg Khella extract 500 mg/Kg Khella extract DayPercent survival Figure 4-1. Survival rate of an imal after treatment with KE.

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68 0 50 100 150 200Control 0.75% EG + NH4Cl 2.5 g /kg Potassium citrate 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract U/L Figure 4-2. Lactate dehydrogena se in urine at day 7 (U/L ) after treatment with KE. 0 25 50 75 100Control 0.75% EG + NH4Cl 2.5 g /kg Potassium citrate 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract U/L Figure 4-3. Alkaline phosphatase in urine at day 7 (U/L) after treatment with KE.

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69 0 5 10 152.5 g /kg Potassium citrate Control 0.75% EG + NH4Cl 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract mL Figure 4-4. Urine volume at day 7 (mL) after treatment with KE. 0.0 0.5 1.0 1.5 2.0Control 0.75% EG + NH4Cl 2.5 g /kg Potassium citrate 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract mg/day Figure 4-5. Urinary calcium excretion at day 7 (mg/d) after treatment with KE.

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70 0.0 0.5 1.0 1.5 2.0 2.5125 mg/kg Khella extract Control 0.75% EG + NH4Cl 2.5 g /kg Potassium citrate 250 mg/kg Khella extract 500 mg/kg Khella extract *mg/day Figure 4-6. Urinary oxalate excret ion at day 7 (mg/d) after treat ment with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001). 0.0 2.5 5.0 7.5 10.0Control 0.75% EG + NH4Cl 2.5 g /kg Potassium citrate 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract ** ** **mg/day Figure 4-7. Urinary citrate excr etion at day 7 (mg/d) after tr eatment with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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71 0 5 5.5 6.0 6.5 7.0Control 0.75% EG + NH4Cl 2.5 g /kg Potassium citrate 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract *pH Figure 4-8. Urine pH after treatment w ith KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001). 0 1 2 3 42.5 g /kg Potassium citrate Control 0.75% EG + NH4Cl 125 mg/kg Khella extract 250 mg/kg Khella extract 500 mg/kg Khella extract ** ** *CaOX crystal depositions score Figure 4-9. Calcium oxalate crys tal deposition score, <1= 0, <10 = 1, <30 = 2, <50 = 3, < 75 = 4 and 75 = 5 after treatment with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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72 Control EG+NH4Cl PCi(2.5 g/kg) Khella125 mg/kg 250 mg/kg 500 mg/kg Figure 4-10. Histopathology of rat kidney (arrows pointed at the calcium oxalate crystals).

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73 Day7 0.0 0.5 1.0 1.5 2.0 2.5 3.0mg/day Day14 0.0 0.5 1.0 1.5 2.0 2.5 3.0Control 0.75% EG + 1.0% NH4Cl 2.5 g/kg PCi 5 mg/kg Khellin 10mg/kg Khellin 5 mg/kg Visnagin 10mg/kg Visnagin mg/day Figure 4-11. Urinary calcium excretion (mg/d) at day 7 and day 14 after treatment with khellin and visnagin (comparing to 0.75 % EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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74 0.0 0.5 1.0 1.5 2.0 2.5Day 7mg/day 0.0 0.5 1.0 1.5 2.0 2.5Control 0.75% EG + 1.0% NH4Cl 2.5 g/kg PCi 5 mg/kg Khellin 10mg/kg Khellin 5 mg/kg Visnagin 10mg/kg Visnagin Day 14mg/day Figure 4-12. Urinary oxalate excretion (mg/ d) at day 7 and day 14 after treatment wi th khellin and visnagin (comparing to 0.75 % EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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75 0 1 2 3 4 5*Day 7mg/day 0 1 2 3 4 5Control 0.75% EG + 1.0% NH4Cl 2.5 g/kg PCi 5 mg/kg Khellin 10mg/kg Khellin 5 mg/kg Visnagin 10mg/kg Visnagin Day 14mg/day Figure 4-13. Urinary citrate excretion (mg/ d) at day 7 and day 14 after treatment w ith khellin and visnagin (comparing to 0.75 % EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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76 0 5 5.5 6.0 6.5 7.0*Day 7pH 0 5 5.5 6.0 6.5 7.0*Control 0.75% EG + 1.0% NH4Cl 2.5 g/kg PCi 5 mg/kg Khellin 10mg/kg Khellin 5 mg/kg Visnagin 10mg/kg Visnagin Day 14pH Figure 4-14. Urinary pH excretion (mg/d) at day 7 and day 14 after treatment with khe llin and visnagin (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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77 0 1 2 3 45 mg/kg Khellin Control 0.75% EG + 1.0% NH4Cl 2.5 g/kg PCi 10mg/kg Khellin 5 mg/kg Visnagin 10mg/kg Visnagin * *CaOX crystal depositions score Figure 4-15. Calcium oxalate cr ystal deposition score, <1= 0, <10 = 1, <30 = 2, <50 = 3, < 75 = 4 and 75 = 5 after treatment with KE (comparing to 0.75% EG +1.0% NH4Cl, p < 0.5, ** p< 0.01, *** p< 0.001).

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78 Figure 4-16. Handling of citrate by hypothetical renal tubule(81).

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79 Figure 4-17. Potential mechanism of increased urinary citrate with systemic alkalosis(82).

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80 CHAPTER 5 PHARMACOKINETIC ANALYSIS OF VISNAGIN IN RATS Background Visnagin (Figure 5-1A) is a furanocoum arin de rivative and one of the major constituents in Ammi visnaga L. (Khella, apiaceae). Khella was used by ancient Egyptians as a treatment for kidney stone disease. Visnagin has cardiovascular effects due to calcium channel blocking actions (33, 36). It can inhibit va scular smooth muscle contraction and seems to dilate peripheral and coronary vessels and increase coronary circulation (33, 36, 38-40). Visnagin also has negative chronotropic and inotropi c effects and reduces peripheral vascular resistance (33, 41). Khella extracts appear to have some antimicrobial activity; this might be attributable to both the khellin and visnagin constituents, which both seem to have antifungal, antibacterial, and antiviral activity (34). Our previous study showed that the preventive effect of urolithiasis (kidney stone formation) in cell culture and animal experime nt (97, 98). In addition, khellin and visnagin decreased calcium oxalate crystal growth in rats as well. Although, visnagin has been studied for its therapeutic use for more than 10 years, th ere are still no information available about its pharmacokinetics Various analytical methods have been used to identify and quantify visnagin, including thin-layer chromatography (TLC) (99, 100), HPLC with solid-phase extraction (SPE) (101, 102), capillary electrophoresis (103) and high perfor mance liquid chromatography (HPLC) (55, 56, 58, 59, 104-107). All of these methods involved multi-st ep extractions, large volume of organic solvent and high volume of sample. In order to fully characterize the pharmacokinetics of visnagin, we wanted to collect blood samples in each animal at multiple time points, which limits the per sample blood volume (not more than 10 % of total blood volume). Therefore, a simple,

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81 sensitive, and robust method with small samp le volume requirements was needed and the pharmacokinetics in rats treated with oral and intravenous administration of visnagin were performed to obtain information about its absorption and disposition.. Specific Aim The purpose of this study was to develop a sensitive and highly selective m ethod based on liquid chromatography-tandem mass spectrometry (LCMS) to determine visnagin in rat plasma and to obtain the pharmacokine tic profile of visnagin in rat. Materials and Methods Chemicals Visnagin (>97%), warfarin (98%) and polyethelene glycol 200 (PEG 200) were purchased from Sigma (St. Louis, MO, USA). All chemicals used in the study were analytical grade. Ammonium acetate, formic acid (88%), and acetonitrile were obtained from VWR (West Chester, PA, USA). Methanol was purchased fr om Thermo Fisher Scientific (Pittsburgh, PA, USA). HPLC grade deionized water was prep ared using a Barnstead Nanopure Diamond UV ultra pure Water System (Dubuque, IA, USA). Liquid Chromatography/Tendem Mass Spectrometry (L C-MS/MS)Analysis Stock, work solution, and prepar ation of calib ration standards Stock solutions of visnagin 0.5 mg/mL and wa rfarin (internal standard) 5000 ng/mL were prepared in methanol and kept in 4 C. Visnagin stock solution: 0.5 mg/mL (500,000ng/mL): visnagin 5 mg was accurately weighed and transferred to 10 mL volumetric flask. The standard was then dissolved in methanol and the volume was completed with same solvent. Warfarin stock solution : 5,000 ng/mL: warfain 5 mg was accurately weighed and transferred to 10 mL volumetric flask. The standa rd was then dissolved in methanol and the

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82 volume was completed with same solvent. 1 mL of 0.5 mg/mL was transferred to 100 mL volumetric flask and the volume was completed with methanol. Wafarin was used as an internal standard (Figure 5-1B). Visnagin work solution: From the visnagin stock solution, fi ve different concentrations of work solutions were prepared in methanol by di luting visnagin stock so lution according Table 51 to the 1.5 mL centrifuge tube and adding methanol to 1 mL total volume. Standard solution of visnagin plasma : From the visnagin working solution, seven different concentration of the standard solution a nd three quality controls (QC) were prepared in methanol and then spiked to plasma according table 5-2. LC-MS/MS condition The LC-MS/MS system consisted of a Surveyor HPLC autosampler, Surveyor MS quaternary pump and a TSQ Quantum Disc overy triple quadrupole mass spectrometer (ThermoFisher Scientific, San Jose, CA, US A). The TSQ Quantum was equipped with an electrospray (ESI) ionization s ource and operated in the positive ion mode. The ESI source spray was set orthogonal to the ion transfer tube.The MS/MS conditions were optimized by infusing visnagin in methanol while the ESI source pa rameters were tuned for maximum abundance of [M+H]+ ions of visnagin at the LC flow rate of 0.2 mL/min. For qua ntification, the TSQ Quantum was operated in high-resolution sele cted reaction monitoring mode (SRM). The instrument parameters included an io n transfer tube temperature of 325oC, spray voltage of 5.0kV and source CID set to 5V. Nitrogen was used as the sheath and auxili ary gas and set to 35 and 10 (arbitrary units), respect ively. The collision energy was 27 eV for visnagin and warfarin and the collision gas (argon) pressure was set to 1.5 mTorr. The SRM scheme followed transitions of the [M+H]+ precursor to selected product i ons with the following values: m/z 231 216 for visnagin and m/z 309 163 for warfarin. The instrument was operated in

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83 enhanced (high) resoluti on mode with the peak width (full width at half maximum, FWHM) set to 0.2 m/z at Q1 and to 0.70 m/z at Q3. Chromatography was performed using Phenom enex Synergi Max RP, 75mm x 2.0 mm ID, 4 m analytical column (Torrance, CA, USA) at am bient temperature. The mobile phase used for analysis was 0.1% formic acid, 5 mM ammonium acetate in deionized water and methanol (15:85, v/v) delivered at a flow rate of 0.2 mL/min. The mobile phase was degassed and filtered through 0.45 m Nylon 66 membrane before use. The autosampler rinse solution was comprised of isopropanol, acetronitrile, and water (35:35:30, v/v/v) containing 0.1% formic acid. Data were acquired and processed using ThermoFinnigan XCa libur software version 1.4, service release 1 (ThermoFisher San Jose, CA, USA). Method validation Calibration curves were construc ted by linear regression of the peak area ratio of visnagin to warf arin ( Y -axis) and the nominal standa rd visnagin concentration (X -axis) with a weighting factor of 1/y2. Concentrations of QCs and samples we re calculated by using the regression equation of the calibration curve. Standards at all concentrati on were analyzed in duplicate. The method was validated with respect to selectivity, carry over linearity, precision, accuracy, extraction recovery and matrix effect (108). Carry over was evaluated by placing vials of methanol at several locations in the analysis set. The accuracy and precision of the assay was determined by the analysis of QC samples at visnagin concentrations of 20, 850 and 4,000 ng/mL. Replicate QC samples (n=8) were analyzed in each of three runs to determine intra-run and inter-run precision and accuracy. Precision is presented as percent relative standard deviation values (R.S.D.%), which were calculated using one-way ANOVA usi ng run as the grouping variable. Accuracy was calculated as the per cent error in the calculated mean concentration

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84 relative to the nominal visnagin concentrati on (R.E.%). For the assa y to be considered acceptable, the precision and accuracy determined at each QC concentration level was required to be within 15%. The ex traction recovery and absolute matrix effect were evaluated for visnagin samples prepared at concentrations of 20 and 4000 ng/mL and for the internal standard warfarin at a concentration of 500 ng/mL. Each set of sa mples was analyzed in triplicate. Extraction recovery was determined by comparing peak areas of the visnagin from spiked matrix (blank plasma containing visnagin) in the same manne r and spiked after extr action with the same standard concentration. Matrix effect on ionization was evalua ted by comparing the visnagin peak areas of samples spiked post-extraction wi th corresponding peak area ratios of visnagin standards prepared in the injection solution. Animals Experiment Male Sprague-Dawley rats (weighing 250 g, n= 8) purchased from Harlan (Indianapolis, IN, USA) were used for the oral study and Male Sprague-Dawley rats with a catheter inserted in the jugular vein, (weighing 250 g, n=8) were obtained from Charles River Laboratories (Wilmington, MA, USA) and used for the IV st udy. The animals were housed in plastic cages and were allowed to adapt to their environment fo r one week before being used for experiments. All the animals were maintained on 12hr/12hr light /dark cycle. They received a standard chow and water ad libitum during the study. A ll animal experiments were performed according to the policies and guidelines of the Institutional An imal Care and Use Committee (IACUC) of the University of Florida, Gainesvill e, USA (NIH publication #85-23). For the pharmacokinetics study, visnagin (10 mg /kg in 2% ethanol and 2% PEG 200) was administered orally by gavage or was injected through the intravenous catheter (1.0 mg/kg in 2% ethanol and 2 % PEG 200) Plasma samples (300 l per blood sample) were collected from the

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85 sublingual vein into Vaccuette heparinized tubes at 0, 10, 20 30 min, 1, 4, 6, 9 and 12 hr for oral administration and at 0, 3, 5, 10, 30 min, 1, 4, 6 and 8 hr for IV injection. Before blood collection, the rats were anaesthet ized with halothane and the blood loss was replaced with 1 mL of normal saline. The blood sample was centrifuged for 15 min at 4,000 rpm at 4 C. The supernatant was transferred in to tubes and stored at -20 C until analysis. Analytical Methods Sample preparation Protein precipitation solvent was prepared by adding 5,000 ng/m L of warfarin to 25:75 MeOH:ACN to get the concentrat ion of 500 ng/mL warfarin. 50 l of plasma was transfered to 1.5 mL centrifuge tubes, then added 200 L of protein precipitation solvent, mixed by vortex for 1 min, next centrifuge at 10,000 rpm for 15 min. Take 100 l of supernatent into 1.5 mL centrifuge tube and dilute with 500 L of DI water, then mixed by vortex for 1 min. Finally, transfer the samples to the LC/MS vial. Blank, st andard and QC solution were also prepared as the same way. Statistical analysis W inNonlin software package, version 5.0, (P harsight Corporation, USA) was used for pharmacokinetic analysis and GraphPad Prism 5 was used for statistical analysis. Data are given as mean with corresponding standard deviation. Results Method validation Figure 5-2 represents the ion chrom atograms of plasma samples. Figure 5-1A shows a blank plasma sample (no visnagin or warfarin ), Figure 5-1B is a limit of quantitation (LOQ) standard visnagin (1 ng/mL), Fig. 5-1C shows a plasma sample obtained 1 hour after intravenous

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86 administration (concentration = 52.9 ng/mL) and Fig. 2D depicts the internal standard warfarin (500 ng/mL). The retention times were approxim ately 1.6 and 1.9 min for vi snagin and warfarin, respectively. The peak of visnagin and warfarin were well separated without the interference of endogenous substance. During method development, we discovered that it was important for visnagin and warfarin to be chromatographically separated because visnagin affected warfarin ionization such that there was an inverse rela tionship between the visnagin concentration and warfarin response. Validation of the assay method was conducted according to United States Food and Drug Administration (FDA) guidelines wi th respect to sele ctivity, carry over, linearity, precision, accuracy. The calibration curve wa s linear over the concentrati on range of 1.0-5000 ng/mL with a mean correlation coefficient (R2) of 0.9922 0.0008. Precision was represented as the relative standard deviation (%RSD) and accuracy was calculated as the relative error (%RE) from the respective nominal concentration. The maximum acceptable limit for precision and accuracy was set at 15%. The intra-run and in ter-run precision (R.S.D.%) was 4.5% and accuracy (R.E.%) was 4.3% (Table 5-3). There was no evidence of sample carry-over. In addition, the matrix effect assessed by spiking samples post-pro cessing showed <10% difference from spiked injection solvent. The mean extraction recoveri es (n=6) for visnagin were 100.8 %and 100.4% at concentration of 20 and 4000 ng/mL, respectively. The extraction recovery for warfarin was 107.1%. Pharmacokinetic Study of Visnagin The plasm a concentration-time profile and pha rmacokinetic parameters of visnagin after oral and intravenous administrations were show n in Figure 5-3 and Table 5-4, respectively. For oral administration, plasma concentration of visnagin reached the maximum level of 3270.72

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87 ng/mL at 0.33 h and decreased to below lim it of quantitation (1.0 ng/mL) after 12 h. The bioavailability (F) of visnagin was 100.71% at does 10 mg meaning the completely absorption. For intravenous administration, the maximum concentration of visnagin was 1635.76 ng/mL at 0 h. Discussion and Conclusion Method Validation The applic ation of LC-MS/MS for analysis of vi snagin in rat plasma resulted in a sensitive, simple and robust method. The method was developed and validated with re spect to selectivity, carry over, linearity, precision and accuracy and requires only a small amount of rat plasma (50 L). Therefore, the present method is applicable to study the pharmacokinetics of visnagin in rat plasma. Pharmacokinetic Study of Visnagin Non com partmental analysis reveal ed that after oral administra tion of visnagin at a dose of 10 mg/kg (in 2% ethanol and 10% PEG 200), the compound was completely absorbed (oral bioavailability, F=100.71%). The half-lives of 0.79 and 0.61 h from oral and intravenous administration were short. The volume of distribution (Vd) of visnagin was 0.86 L, which is suggestive of the distribu tion into extracellular fluids in the body (109). The plasma volume of a rat weighing 250g is approximately 7.8 mL (110). To our knowledge, this is the first pharmacokinetic study of visnagin, thus comparable data are in lacking. However, the complete absorption of visnagin from this study could be overestimated. It might be that visnagin has a short half-life and the blood sampling time at th e early time points have not been sufficient. Therefore, the area under the curve after intrav enous administration was underestimated and as a result the bioavailability was elevated. Anothe r reason is that the capacity-limited metabolism could happen. The capacity-limited metabolism is al so called saturable metabolism (111). First,

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88 the drugs interact with enzyme to produce a drug-enzyme intermediate. Then intermediate complex is further processed to produce metabolites and release the enzyme. The released enzyme is recycled and reacts with more drug mo lecules. Based on this relationship, at very low drug concentrations, the concentrat ion of available enzyme is mu ch larger than number of drug molecules. Therefore, when the concentration of the drug is incr eased, the rate of metabolism is increased almost proportionally. Howe ver, after certain points, as the concentration increases the rate of metabolism increases less than propor tional. The other extreme occurs when the concentration of the drug is very high relativ e to the concentration of available enzyme molecules. Under this condition, a ll of the enzymes are saturated with the drug molecules (111). This would allow a disproportionate fraction of the absorbed drug to escape into the systemic circulation and result in elevat ed plasma levels and therefore increased AUCs following the oral dose (111-113). Recently, pharmacokinetic studies of khellin in rats have been performed and the authors reported an oral bioavilability of 5.66 and 6.77% in the dose of 1.25 and 2.5 mg when khellin was administered as a tablet (114). Although khellin and visn agin are both furanocoumarin derivatives (Figure 1-6), our study showed a different oral bioavailability of visnagin (F=100.71%). Khellin pharmacokinetics could be fitted to a two compartment open model after oral administration in rats and rabbits (114, 115). However, our da ta from oral and intravenous administration of visnagin could not be fitted with a two compartment model. One possible reason could be that the sampling of the later ti me points was not sufficien t. Figure 5-3 displays the plasma concentration-time profile of visnag in after oral and intr avenous administration demonstrating that the elimination phase could be biexponential. In order to fully answer their question, more frequent bl ood samplings are necessary.

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89 The present results suggest that the sampling time in this study might not sufficient to obtain the correct bioavailability value and to de termine the compartmenta l analysis. In addition, more doses on a lower concentration as well as urin e analysis would be of great interest to obtain a full pharmacokinetic profile. However, the resu lts from this experiment are a good start for studying the pharmacokinetics of visnagin a nd further furanocoumarin derivatives.

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90 Table 5-1. concentrations of working soluti on used for preparing the standard solution Working solution Volume (mL) From MeOH (mL) Working solution concentration (ng/mL) 1 0.2 Stock solution 0.8 100,000 2 0.5 Working solution 1 0.5 50,000 3 0.2 Working solution 2 0.8 10,000 4 0.4 Working solution 3 0.6 4,000 5 0.1 Working solution 3 0.9 1,000 6 0.1 Working solution 4 0.9 100

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91 Table 5-2. Concentrations of standard soluti on used for the calibration curves and quality control Standard Standard concentration (ng/mL) Plasma standard Volume (mL) Working solution (ng/mL) Spiking amount ( l) in plasma 1 0.5 1 100 5 2 1 1 100 10 3 10 1 1,000 10 4 100 1 10,000 10 5 500 1 50,000 10 6 2500 1 500,000 5 7 5000 1 500,000 8 QC 1 20 1 4,000 5 QC 2 850 1 100,000 8.5 QC 3 4000 1 500,000 10

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92 Table 5-3. Intra-day (n=8) and inter-day (n =24) precision (%CV) a nd accuracy (%RE) for analysis of visnagin in blank rat plasma. Nominal concentration (ng/mL) HC LC MC Characteristic Statistic 4,000 20 850 # Results N 24 24 24 Accuracy Mean Bias (RE%) 4.3 2.9 3.5 Precision Intra-run (RSD%) 3.1 3.6 4 Inter-run (RSD%) 4.4 4.5 4.3

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93 Table 5-4. Pharmacokinetic parameters from mean plasma conetration-time profile using noncompartmental analysis after oral and intravenous administration (n=8). Parameter Oral IV Dose (mg/kg) 10 1 AUC0-last (ng*h/L) 10.36 1.03 AUC0(ng*h/L) 10.37 1.03 Cl/F (L/h/kg) 0.96 Vd/F(L/kg) 1.10 Cl (L/h/kg) 0.97 Vd(L/kg) 0.86 Tmax (h) 0.33 Cmax (ng/mL) 3270.72 Ke (1/h) 0.88 1.13 t1/2 (h) 0.79 0.61 C0 (ng/mL) 1635.76 F(%) 100.71

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94 Figure 5-1. Structures of (A) visnagin and (B) warfarin (i nternal standard).

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95 0.0 0.4 0.8 1.2 1.6 2.0 2.4 0 250 500 750 1000(A) Visnagin ( m/z : 231 > 216)Intensity (cps) 0.0 0.4 0.8 1.2 1.6 2.0 2.4 0 6000 12000 18000RT: 1.59(B) Visnagin ( m/z : 231 > 216)Intensity (cps) 0.0 0.4 0.8 1.2 1.6 2.0 2.4 0 100000 200000 300000 400000RT: 1.59(C) Visnagin ( m/z : 231 > 216)Intensity (cps) 0.0 0.4 0.8 1.2 1.6 2.0 2.4 0 100000 200000 300000 400000 500000RT: 1.86(D) Warfarin (IS) ( m/z : 309 > 163)Time (min)Intensity (cps) Figure 5-2. The extracted LC-MS/MS chromatogram s. A) Blank rat plasma. B) Spiked lowest standard visnagin.(limit of quantitation, 1ng/ mL). C) Plasma sample from a rat obtained 1.0 h after intravenous administ ration of visnagin (concentration = 52.9 ng/mL). D) warfarin (500 ng/mL) as an internal standard.

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96 (A) 0.0 0.5 0 1000 2000 3000 4000 5000 2 4 6 8 10 12 Time (hr)[visnagin] (ng/mL) (B) 0 1 0 500 1000 1500 2000 2500 2 4 6 8 Time (hr)[visnagin] (ng/mL) 0 2 4 6 8 10 12 14 0.01 0.1 1 10 100 1000 10000 (C)Time (hr)log [visnagin] (ng/mL) 0 2 4 6 8 10 0.01 0.1 1 10 100 1000 10000 (D)Time (hr)log [visnagin] (ng/mL) Figure 5-3. Plasma concentration-time curve. A) Oral administration (10 mg/kg). B) Intravenous administration (1 mg/kg). C) Oral administration plotted as log scale. D) Intravenous administration plotted as log scale. Error ba rs refers to the standard deviation of concentration data at each sampling time point (n=8).

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97 CHAPTER 6 CONCLUSION Kidney ston e disease is a comm on urinary system disorder in humans and often causes severe pain, which may lead to emergent hospita lization, shock wave lithotripsy, and/or surgery. Calcium oxalate is a major component if the ki dney stone. It forms upon the supersaturation of the urine with calcium and other salt, especially oxalate. The size of the stone can increase and obstruct in the urinary system. Current pharmacol ogical treatment of kidn ey stones consists of alkalosis supplement such as pot assium citrate. Although the most effective treatment of kidney stone is extracorporeal shock wave lithotripsy, the side effects of this method are grave and can lead to recurrence of kidney stones. Therefore, alternative treatments are of high interest. Khella ( Ammi visnaga L.) has long been used by Egyptia ns to treat kidney stones. The major constituents in Khella are coumarins incl uding khellin and visnagin. Khellin acts as a spasmolytic agent and visnagin also may reduce peripheral vascular resistence which may help the stone easily pass through the urinary system harmlessly. In this study, KE and its major components, khellin and visnagin, showed preventive activity with in vitro cell culture experiment ation and decreased lact ate dehydrogenase enzyme. In addition, KE, khellin and visnagin also showed the preventive effect in vivo by decreasing calcium oxalate crystal deposition. The mechanis m of action is unknown. It may be explained by changes in urinary pH that interfered with ci trate reabsorption in the kidney, consequently the citrate inhibits the formation and aggregation of calcium oxalate stones in the urinary system. Additionally, khellin and visnagin may aid in pass age of small stones thro ugh the urinary tract. The pharmacokinetic study revealed that visnag in was a short half-l ife compound with 0.79 and 0.61 h after oral and intravenous administration, respectively. The oral bioavailability was

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98 100.71% meaning the completely ab sorption. In conclusion, our data suggest KE could be used as a potential therapeutic strategy in the pr evention of kidney stones caused by hyperoxaluria

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99 APPENDIX A ADDITIONAL TABLES Cell Culture Experiment Table A-1. The % LDH release from oxalate ex posure to L LC-PK1 with or without KE (N=6) 1 hour 3 hour 6 hour MeanStd. Dev MeanStd. Dev Mean Std. Dev Control 6.1800.906 6.6320.630 7.474 0.778 300 M Ox 6.8360.456 8.4560.762 9.096 0.061 300 M Ox + 10 g KE 6.9791.307 8.0370.661 6.874 0.756 300 M Ox + 50 g KE 7.4751.387 7.9071.235 6.445 0.886 300 M Ox + 100 g KE 5.0950.537 5.4150.941 5.456 0.309 300 M Ox + 200 g KEt 5.6150.652 6.4301.114 5.742 0.384 Table A-2. The % LDH release from calcium ox alate crystals exposure to LLC-PK1 with or without KE (N=6) 1 hour 3 hour 6 hour MeanStd. Dev MeanStd. Dev Mean Std. Dev Control 7.0070.477 7.3371.375 6.963 0.715 133 g/cm2 COM 7.5581.018 8.1090.903 13.258 1.075 133 g/cm2 COM+ 10 g KE 6.9410.914 7.7460.729 12.659 1.383 133 g/cm2 COM+ 50 g KE 6.2110.634 6.3900.546 10.571 0.632 133 g/cm2 COM+ 100 g KE 6.0230.913 6.6320.486 10.204 1.328 133 g/cm2 COM+ 200 g KE 5.6640.648 5.9240.471 8.826 0.394 Table A-3. The % LDH release from oxalate ex posure to MDCK with or without KE (N=6) 1 hour 3 hour 6 hour Mean Std. DevMean Std. Dev Mean Std. Dev Control 11.8151.314 18.9241.672 15.5391.357 300 M Ox 31.1854.062 30.9356.571 25.4136.841 300 M Ox + 10 g KE 34.5634.301 33.7266.538 27.3467.157 300 M Ox + 50 g KE 34.1816.064 36.0986.897 34.3593.905 300 M Ox + 100 g KE 22.9638.365 17.5132.503 21.2426.112 300 M Ox + 200 g KE 21.0675.335 25.5424.170 24.5395.787

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100 Table A-4. The % LDH release from calcium ox alate crystals exposure to MDCK with or without KE(N=6) 1 hour 3 hour 6 hour MeanStd. Dev MeanStd. Dev Mean Std. Dev Control 2.1710.374 2.9990.744 3.224 1.305 133 g/cm2 COM 4.6200.860 5.0400.598 6.715 1.031 133 g/cm2 COM+ 10 g KE 5.3840.520 6.5730.746 8.767 1.048 133 g/cm2 COM+ 50 g KE 6.6120.848 7.3031.753 11.991 3.203 133 g/cm2 COM+ 100 g KE 2.0040.661 2.4480.651 3.952 0.876 133 g/cm2 COM+ 200 g KEt 3.3171.228 4.0870.751 5.149 0.659 Table A-5. The % LDH release from oxalate expos ure to LLC-PK1 with or without khellin or visnagin (N=6) 1 hour 3 hour 6 hour MeanStd. Dev MeanStd. Dev Mean Std. Dev Control 4.1550.775 5.2891.328 4.488 1.768 300 M Ox 7.5780.280 8.5980.799 14.759 1.851 300 M Ox + 100 g KE 5.6790.739 6.1550.664 7.459 0.655 300 M Ox + 2.81 g Khellin 6.5191.530 7.0981.003 8.163 1.523 300 M Ox + 1.72 g Visnagin 5.6040.377 5.7820.593 5.806 1.306 Table A-6. The % LDH release from calcium ox alate crystals exposure to LLC-PK1 with or without khellin or visnagin (N=6) 1 hour 3 hour 6 hour MeanStd. Dev Mean Std. Dev Mean Std. Dev Control 4.1550.775 5.289 1.328 4.488 1.768 133 g/cm2 COM 9.0520.591 12.8131.032 15.957 1.976 133 g/cm2 COM+ 100 g KE 8.3670.555 9.798 1.271 12.194 1.447 133 g/cm2 COM+ 2.81 g Khellin 6.7920.907 8.943 1.027 12.080 1.576 133 g/cm2 COM+ 1.72 g Visnagin 6.3870.955 6.119 0.653 10.693 1.412

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101 Table A-7. The % LDH release from oxalate ex posure to MDCK with or without khellin or visnagin (N=6) 1 hour 3 hour 6 hour Mean Std. DevMean Std. Dev Mean Std. Dev Control 8.040 0.511 7.975 0.232 12.8031.289 300 M Ox 14.8531.696 14.4332.567 17.4532.437 300 M Ox + 100 g KE 10.5212.854 11.7480.725 14.2163.477 300 M Ox + 2.81 g Khellin 14.2051.951 12.8791.814 13.8951.162 300 M Ox + 1.72 g Visnagin 16.4463.889 15.5623.583 17.2323.424 Table A-8. The % LDH release from calcium ox alate crystals exposure to MDCK with or without khellin or visnagin (N=6) 1 hour 3 hour 6 hour Mean Std. DevMean Std. Dev Mean Std. Dev Control 8.040 0.511 7.975 0.232 12.8031.289 133 g/cm2 COM 16.9631.838 18.5241.370 18.1131.440 133 g/cm2 COM+ 100 g KE 12.0480.587 15.0521.763 12.8081.726 133 g/cm2 COM+ 2.81 g Khellin 15.3621.719 19.8884.860 15.8351.916 133 g/cm2 COM+ 1.72 g Visnagin 14.4441.914 16.3796.960 12.4351.966

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102 Animal Experiment Table A-9. Lactate dehydrogenase (LDH)and alkaline phosphatase (ALP) in urine (U/L) at day 0 after treatm ent with KE # of values LDH (U/L) ALP (U/L) MeanStd. Dev Mean Std. Dev Control 8 28.3 6.4 67.5 24.1 0.75% EG + NH4Cl 8 34.5 16.3 70.8 13.6 2.5 g /kg Potassium citrate 8 31.3 7.8 66.8 19.9 125 mg/kg KE 8 30.3 4.7 70.5 18.8 250 mg/kg KE 8 29.5 5.1 69.5 22.3 500 mg/kg KE 8 33.3 7.2 67.5 23.4 Table A-10. Lactate dehydrogena se (LDH) and alkaline phosphata se (ALP) in urine (U/L) at day 7 after treatment with KE # of values LDH (U/L) ALP (U/L) MeanStd. Dev Mean Std. Dev Control 8 33.0 8.0 68.5 21.4 0.75% EG + NH4Cl 8 92.3 52.2 98.5 42.8 2.5 g /kg Potassium citrate 8 83.5 14.1 76.5 19.2 125 mg/kg KE 8 128.551.1 86.3 28.1 250 mg/kg KE 8 90.3 28.6 95.0 40.1 500 mg/kg KE 8 111.339.3 60.8 24.4 Table A-11. Lactate dehydrogenase (LDH)and alkaline phosphatase (ALP) in urine (U/L) at day 14 after treatment with KE # of values LDH (U/L) ALP (U/L) MeanStd. Dev Mean Std. Dev Control 8 39.0 6.0 66.3 26.1 0.75% EG + NH4Cl 2 117.09.9 100.0 11.3 2.5 g /kg Potassium citrate 8 84.6 30.7 53.1 22.6 125 mg/kg KE 8 119.330.2 61.3 16.1 250 mg/kg KE 8 113.338.5 60.5 22.9 500 mg/kg KE 7 128.947.6 61.4 18.8

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103 Table A-12. Calcium (Ca), citrate, oxalate (Ox) in urine (U/L) at da y 0 after treatment with KE # of values Ca (mg/day) Citrate (mg/day) Ox (mg/day) pH MeanSTDMeanSTDMeanSTD Mean STD Control 8 1.34 0.62 1.36 0.30 0.42 0.18 6.46 0.15 0.75% EG + NH4Cl 8 1.25 0.45 1.17 0.31 0.34 0.08 6.26 0.14 2.5 g /kg Potassium citrate 8 1.56 0.51 1.37 0.61 0.46 0.14 6.15 0.11 125 mg/kg Khella extract 8 1.14 0.73 1.31 0.26 0.49 0.16 6.28 0.17 250 mg/kg Khella extract 8 1.50 0.48 1.55 0.32 0.49 0.11 6.15 0.11 500 mg/kg Khella extract 8 1.43 0.33 1.60 0.41 0.54 0.07 6.08 0.14 Table A-13. Calcium (Ca), citrate, oxalate (Ox) in urine (U/L) at day 7 after treatment with KE # of values Ca (mg/day) Citrate (mg/day) Ox (mg/day) pH MeanSTDMeanSTDMeanSTD Mean STD Control 8 1.21 0.40 1.38 0.27 0.45 0.05 6.03 0.11 0.75% EG + NH4Cl 8 0.64 0.26 1.10 0.21 1.01 0.34 5.64 0.10 2.5 g /kg Potassium citrate 8 0.79 0.22 3.16 1.20 1.50 0.39 6.23 0.33 125 mg/kg Khella extract 8 0.74 0.36 4.00 0.67 0.79 0.34 5.84 0.17 250 mg/kg Khella extract 8 0.94 0.17 4.02 0.34 0.58 0.22 5.91 0.18 500 mg/kg Khella extract 8 0.91 0.41 5.85 2.45 0.57 0.15 6.05 0.15

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104 Table A-14. Calcium (Ca), citrate, oxalate (Ox) in urine (U/L) at day 14 after treatment with KE # of values Ca (mg/day) Citrate (mg/day) Ox (mg/day) pH MeanSTDMeanSTDMeanSTD Mean STD Control 8 1.10 0.25 1.69 0.48 0.66 0.09 6.18 0.19 0.75% EG + NH4Cl 2 0.79 0.08 1.06 0.04 0.98 0.01 6.55 0.01 2.5 g /kg Potassium citrate 8 1.21 0.46 2.67 1.09 1.82 0.86 6.63 0.27 125 mg/kg Khella extract 8 1.19 0.39 4.00 2.09 1.05 0.85 6.10 0.25 250 mg/kg Khella extract 8 1.33 0.67 4.07 1.45 0.68 0.32 6.18 0.24 500 mg/kg Khella extract 7 1.21 0.32 5.31 1.33 0.73 0.17 5.99 0.15 Table A-15. Calcium oxalate crysta l deposition and score in kidney # of values CaOx crystal deposition CaOx crystal deposition score Mean Std. Dev. Mean Std. Dev. Control 8 0.0 0.00 0.00 0.00 0.75% EG + NH4Cl 8 44.4 22.21 3.13 1.25 2.5 g /kg Potassium citrate 8 8.5 8.12 1.25 0.71 125 mg/kg Khella extract 8 14.0 15.97 1.63 1.19 250 mg/kg Khella extract 8 19.6 15.95 1.88 0.83 500 mg/kg Khella extract 8 8.5 12.93 1.25 0.71

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105 Table A-16. Calcium (Ca), citrate, oxalate (Ox) in urine (U/L) at day 0 after treatment with khellin or visnagin # of values Ca (mg/day) Citrate (mg/day) Ox (mg/day) pH MeanSTDMeanSTDMeanSTD Mean STD Control 4 1.41 0.35 1.75 0.20 0.32 0.06 6.64 0.10 0.75% EG + NH4Cl 6 1.23 0.39 1.66 0.38 0.33 0.10 6.50 0.24 2.5 g /kg Potassium citrate 6 1.58 0.54 1.88 0.38 0.33 0.06 6.30 0.19 5 mg/kg Khellin 8 1.61 0.71 1.96 0.21 0.40 0.04 6.53 0.18 10 mg/kg Khellin 8 1.30 0.47 1.95 0.39 0.36 0.08 6.45 0.17 5 mg/kg Visnagin 8 1.71 0.56 2.13 0.22 0.38 0.03 6.52 0.20 10 mg/kg Visnagin 8 1.54 0.33 2.12 0.31 0.36 0.05 6.37 0.16 Table A-17. Calcium (Ca), citrate, oxalate (Ox) in urine (U/L) at day 7 after treatment with khellin or visnagin # of values Ca (mg/day) Citrate (mg/day) Ox (mg/day) pH MeanSTDMeanSTDMeanSTD Mean STD Control 4 1.22 0.21 2.07 0.13 0.42 0.06 6.46 0.09 0.75% EG + NH4Cl 6 0.46 0.19 1.40 0.44 0.85 0.29 5.84 0.08 2.5 g /kg Potassium citrate 6 0.71 0.40 2.55 0.22 1.22 0.20 6.27 0.03 5 mg/kg Khellin 8 0.71 0.25 1.58 0.15 0.77 0.19 5.73 0.10 10 mg/kg Khellin 8 0.77 0.57 1.50 0.46 0.77 0.09 5.92 0.25 5 mg/kg Visnagin 8 0.73 0.38 2.03 0.34 1.03 0.40 5.92 0.31 10 mg/kg Visnagin 8 0.57 0.27 1.98 0.86 1.03 0.39 5.86 0.13

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106 Table A-18. Calcium (Ca), citrate, oxalate (Ox) in urine (U/L) at day 14 after treatment with khellin or visnagin # of values Ca (mg/day) Citrate (mg/day) Ox (mg/day) pH MeanSTDMeanSTDMeanSTD Mean STD Control 4 1.39 1.16 2.19 0.43 0.44 0.09 6.40 0.16 0.75% EG + NH4Cl 6 0.55 0.38 1.51 0.89 0.77 0.35 5.96 0.09 2.5 g /kg Potassium citrate 6 1.03 0.65 2.50 0.72 0.85 0.35 6.40 0.36 5 mg/kg Khellin 8 0.72 0.48 1.76 0.37 0.62 0.25 5.97 0.15 10 mg/kg Khellin 8 1.09 0.68 1.99 0.86 0.65 0.31 5.76 0.09 5 mg/kg Visnagin 8 0.89 0.42 1.86 0.37 0.59 0.19 5.81 0.15 10 mg/kg Visnagin 8 1.16 0.68 2.18 0.62 0.68 0.24 5.87 0.12 Table A-19. Calcium oxalate crys tal deposition and score in kidne y after treatmen t with khellin or visnagin # of values CaOx crystal deposition CaOx crystal deposition score Mean Std. Dev. Mean Std. Dev. Control 4 0.00 0.00 0.00 0.00 0.75% EG + NH4Cl 6 42.83 8.01 3.33 0.52 2.5 g /kg Potassium citrate 6 12.67 7.34 1.50 0.55 5 mg/kg Khellin 8 25.25 24.11 2.25 1.28 10 mg/kg Khellin 8 13.75 9.71 1.63 0.52 5 mg/kg Visnagin 8 22.13 20.94 1.88 1.13 10 mg/kg Visnagin 8 9.50 5.78 1.50 0.53

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107 REFERENCES 1. F. L. Coe, A. Evan, and E. Worcester. Kidney stone disease, Journal of Clinical Investigation. 115:2598-2608 (2005). 2. S. R. Khan. Interactions between stone-fo rming calcific crystals and macromolecules, Urologia Internationalis. 59:59-71 (1997). 3. M. Daudon, C. A. Bader, and P. Jungers. Ur inary Calculi Review of Classification Methods and Correlatio ns with Etiology, Scanning Microscopy. 7:1081-1106 (1993). 4. Chris O'Callaghan. The Renal System at a Glance Blackwell Publishing Ltd., Oxaford, UK, 2006. 5. F. C. Delvecchio and G. M. Preminger. Medical management of stone disease, Current Opinion in Urology. 13:229-233 (2003). 6. J.Segura, P. Conort S. Khoury C. Pak G. M. Preminger D. Tolley. Stone Disease. 2003. 2003. Ref Type: Serial (Book,Monograph) 7. Willium F.Kern et al. Atlas of Renal Pathology W.B Saunders Company, USA, 1999. 8. J. A. Jonassen, L. C. Cao, T. Honeyman, and C. R. Scheid. Intracellular events in the initiation of calcium oxalate stones, Nephron Experiment al Nephrology. 98:E61-E64 (2004). 9. S. R. Khan. Renal tubular damage/dysfuncti on: key to the formation of kidney stones, Urological Research. 34:86-91 (2006). 10. R. L. Hackett, P. N. Shevock, and S. R. Khan. Madin-Darby Cani ne Kidney-Cells Are Injured by Exposure to Oxalate and to Calcium-Oxalate Crystals, Urological Research. 22:197-203 (1994). 11. J. D. Valentich. Morphological similaritie s between the dog kidney cell line MDCK and the mammalian cortical collecting tubule, Ann N Y Acad Sci. 372:384-405 (1981). 12. R. Nielsen, H. Birn, S. K. Moestrup, M. Ni elsen, P. Verroust, and E. I. Christensen. Characterization of a kidney proximal tubul e cell line, LLC-PK1, e xpressing endocytotic active megalin, Journal of the American Society of Nephrology. 9:1767-1776 (1998). 13. R. N. Hull, W. R. Cherry, and G. W. Weaver Origin and Characteristics of A Pig Kidney Cell Strain, Llc-Pk, In Vitro-Journal of the Tissue Culture Association. 12:670-677 (1976). 14. S. Yamaguchi, J. H. Wiessner, A. T. Hasega wa, L. Y. Hung, G. S. Mandel, and N. S. Mandel. Study of a rat model for calcium oxala te crystal formation without severe renal damage in selected conditions, International Journal of Urology. 12:290-298 (2005).

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114 99. G. Zgorka, T. Dragan, K. Glowniak, and E. Basiura. Determination of furanochromones and pyranocoumarins in drugs and Ammi visnaga fruits by combined solid-phase extraction-high-performance liquid chromat ography and thin-layer chromatography-highperformance liquid chromatography, Journal of Chromatography A. 797:305-309 (1998). 100. L. Kursinszki, J. Troilina, and E. Szoke. Qu antitative TLC of visnagin in genetically transformed root cultures of Ammi visnaga, Jpc-Journal of Planar ChromatographyModern Tlc. 13:463-467 (2000). 101. L. Kursinszki, J. Troilina, and E. Szoke. Determination of visnagin in Ammi visnaga hairy root cultures using solid-phase extraction and high-performance liquid chromatography, Microchemical Journal. 59:392-398 (1998). 102. G. Zgorka, T. Dragan, K. Glowniak, and E. Basiura. Determination of furanochromones and pyranocoumarins in drugs and Ammi visnaga fruits by combined solid-phase extraction-high-performance liquid chromat ography and thin-layer chromatography-highperformance liquid chromatography, Journal of Chromatography A. 797:305-309 (1998). 103. K. Gunaydin and F. B. Erim. Determination of khellin and visnagin in Ammi visnaga fruits by capillary electrophoresis, Journal of Chromatography A. 954:291-294 (2002). 104. M. M. Eldomiaty. Improved High-Performance Liquid-Chromatographic Determination of Khellin and Visnagin in Ammi-Visnaga Fruits and Pharmaceutical Formulations, Journal of Pharmaceutical Sciences. 81:475-478 (1992). 105. L. Kursinszki, J. Troilina, and E. Szoke. Determination of visnagin in Ammi visnaga hairy root cultures using solid-phase extraction and high-performance liquid chromatography, Microchemical Journal. 59:392-398 (1998). 106. K. Mawatari, S. Mashiko, M. Watanabe, a nd K. Nakagomi. Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using postcolumn photoirradiation, Analytical Sciences. 19:1071-1073 (2003). 107. G. Zgorka, T. Dragan, K. Glowniak, and E. Basiura. Determination of furanochromones and pyranocoumarins in drugs and Ammi visnaga fruits by combined solid-phase extraction-high-performance liquid chromat ography and thin-layer chromatography-highperformance liquid chromatography, Journal of Chromatography A. 797:305-309 (1998). 108. US Department of Health and Human Serv ices Food and Drug Administration. Guidance for Industry: Bioanalytical Method Validati ons, US Department of Health and Human Services. 2001. Beltsville, MD, USA. Ref Type: Patent 109. Michael E.Winter. Basic Clinical Pharmacokinetics Lippincott Williams & Walkins, Baltimore, MD, USA, 2004. 110. B. Davies and T. Morris. Physiological-Par ameters in Laboratory-Animals and Humans, Pharmaceutical Research. 10:1093-1095 (1993).

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116 BIOGRAPHICAL SKETCH Pattaraporn Vanachayangkul grew up in Chiang Rai, the northern m ost region of Thailand. She attended the faculty of pharmacy, Chiang Mai University. In her last year of pharmacy, she recieved a scholarship from the Thailand Resear ch fund awarded for an undergraduate project titled Massage Cream from Lavender Oil. After graduating with a Bachel or of Pharmacy in 2003, she worked for The Olic company, in Thailand, as a production pharmacist in the tabletting department. She began her graduate studies in the Phar maceutics Department at the University of Florida in August 2004. There, she started researching the preventive effect of Ammi visnaga L. in kidney stone diseases under the supe rvision of Dr. Veronika Butterweck.