<%BANNER%>

Xanthine Oxidase Inhibition and Antioxidant Activity of an Artichoke Leaf Extract (Cynara scolymus L.) and its Compounds

Permanent Link: http://ufdc.ufl.edu/UFE0020087/00001

Material Information

Title: Xanthine Oxidase Inhibition and Antioxidant Activity of an Artichoke Leaf Extract (Cynara scolymus L.) and its Compounds
Physical Description: 1 online resource (151 p.)
Language: english
Creator: Sarawek, Sasiporn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: antioxidant, artichoke, cynara, gout, hyperuricemia, luteolin, pharmacokinetics, xanthine
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: Gout is a disease characterized by elevated levels of uric acid in body fluids. This hyperuricemia results in the deposition of urate crystals in tissue, especially joints. The uric acid deposition initiates an inflammation process involving the release of reactive oxygen species. The common treatments of gout are the use of anti-inflammatory agents to relieve the symptoms of the disease and xanthine oxidase (XO) inhibitors to block the synthesis of uric acid. The most common xanthine oxidase inhibitor is allopurinol. However, its use is limited by unwanted side effects such as hypersensitivity problems. Therefore, alternatives are required. Artichoke leaves (Cynara scolymus L.) have been used traditionally by the Eclectic physicians as a diuretic and depurative for the treatment of gout. The major compounds in artichoke leaves are phenolic compounds such as caffeoylquinic acids and flavonoids. These phenolic compounds have shown xanthine oxidase inhibitory activity and antioxidant activity in vitro and in vivo. Therefore, the goal of the present study was to examine the xanthine oxidase inhibitory activity and antioxidant activity of the artichoke extract, and its main compounds in vitro and in vivo. The in vitro study showed that the extract as well as caffeoylquinic acids showed only a weak XO inhibition, whereas flavonoids (flavone and flavonols) had a highly inhibitory effect on XO. Luteolin had the highest XO inhibition effect. This significant inhibition of XO by the flavonoids in vitro suggested that they may suppress the production of uric acid in vivo. However, the in vivo study showed that oral administration of the artichoke extract, caffeoylquinic acids, and flavonoids could not decrease the serum urate levels in oxonate-treated rats. The antioxidant activities of the artichoke extract and its phenolic compounds were determined using the oxygen radical absorbance capacity assay (ORAC). The results showed that the artichoke extract and its compounds elicited an antioxidant activity in vitro, however, the compounds again showed no antioxidant activity in vivo. It was speculated that this lack of effect in vivo from both studies might be due to the absorption, the high first pass effect through intestine and liver, the excretion into urine and bile and the degradation in large intestine. Therefore, the pharmacokinetic of a compound in artichoke was performed in order to explain the in vivo activity. Pharmacokinetic study of luteolin, the compound which showed the highest XO inhibition in vitro, showed that after oral administration of luteolin, luteolin rapidly absorbed and metabolized in plasma. Additionally, plasma-concentration-time curves of luteolin metabolites revealed secondary peaks. The bioavailability of luteolin was low and the urinary excretion of luteolin and its conjugates did not dominate. This study could explain the lack of XO inhibitory activity and antioxidant activity in vivo. Therefore, it can be concluded that artichoke might be not a useful alternative treatment of gout.
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 Sasiporn Sarawek.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Butterweck, Veronika.
Local: Co-adviser: Derendorf, Hartmut C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0020087:00001

Permanent Link: http://ufdc.ufl.edu/UFE0020087/00001

Material Information

Title: Xanthine Oxidase Inhibition and Antioxidant Activity of an Artichoke Leaf Extract (Cynara scolymus L.) and its Compounds
Physical Description: 1 online resource (151 p.)
Language: english
Creator: Sarawek, Sasiporn
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: antioxidant, artichoke, cynara, gout, hyperuricemia, luteolin, pharmacokinetics, xanthine
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: Gout is a disease characterized by elevated levels of uric acid in body fluids. This hyperuricemia results in the deposition of urate crystals in tissue, especially joints. The uric acid deposition initiates an inflammation process involving the release of reactive oxygen species. The common treatments of gout are the use of anti-inflammatory agents to relieve the symptoms of the disease and xanthine oxidase (XO) inhibitors to block the synthesis of uric acid. The most common xanthine oxidase inhibitor is allopurinol. However, its use is limited by unwanted side effects such as hypersensitivity problems. Therefore, alternatives are required. Artichoke leaves (Cynara scolymus L.) have been used traditionally by the Eclectic physicians as a diuretic and depurative for the treatment of gout. The major compounds in artichoke leaves are phenolic compounds such as caffeoylquinic acids and flavonoids. These phenolic compounds have shown xanthine oxidase inhibitory activity and antioxidant activity in vitro and in vivo. Therefore, the goal of the present study was to examine the xanthine oxidase inhibitory activity and antioxidant activity of the artichoke extract, and its main compounds in vitro and in vivo. The in vitro study showed that the extract as well as caffeoylquinic acids showed only a weak XO inhibition, whereas flavonoids (flavone and flavonols) had a highly inhibitory effect on XO. Luteolin had the highest XO inhibition effect. This significant inhibition of XO by the flavonoids in vitro suggested that they may suppress the production of uric acid in vivo. However, the in vivo study showed that oral administration of the artichoke extract, caffeoylquinic acids, and flavonoids could not decrease the serum urate levels in oxonate-treated rats. The antioxidant activities of the artichoke extract and its phenolic compounds were determined using the oxygen radical absorbance capacity assay (ORAC). The results showed that the artichoke extract and its compounds elicited an antioxidant activity in vitro, however, the compounds again showed no antioxidant activity in vivo. It was speculated that this lack of effect in vivo from both studies might be due to the absorption, the high first pass effect through intestine and liver, the excretion into urine and bile and the degradation in large intestine. Therefore, the pharmacokinetic of a compound in artichoke was performed in order to explain the in vivo activity. Pharmacokinetic study of luteolin, the compound which showed the highest XO inhibition in vitro, showed that after oral administration of luteolin, luteolin rapidly absorbed and metabolized in plasma. Additionally, plasma-concentration-time curves of luteolin metabolites revealed secondary peaks. The bioavailability of luteolin was low and the urinary excretion of luteolin and its conjugates did not dominate. This study could explain the lack of XO inhibitory activity and antioxidant activity in vivo. Therefore, it can be concluded that artichoke might be not a useful alternative treatment of gout.
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 Sasiporn Sarawek.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Butterweck, Veronika.
Local: Co-adviser: Derendorf, Hartmut C.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2008-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0020087:00001


This item has the following downloads:


Full Text

PAGE 1

1 XANTHINE OXIDASE INHIBITION A ND ANTIOXIDANT ACTIVITY OF AN ARTICHOKE LEAF EXTRACT ( Cynara scolymus L.) AND ITS COMPOUNDS By SASIPORN SARAWEK 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 2007

PAGE 2

2 2007 by Sasiporn Sarawek

PAGE 3

3 To my parents

PAGE 4

4 ACKNOWLEDGMENTS I would like to express my a ppreciation and very grateful thanks to Dr. Veronika Butterweck for accepting me into her group and for her encouragement and support during my Ph.D. program. My special thanks go to Dr. Hartmut Derendorf for his guidance and helpful advice. Many thanks also go to the member s of my supervisory committee, Dr. Gnther Hochhaus, Dr. Jeffrey Hughes and Dr. Saeed Khan, for their helpful advice throughout the years. I would like to thank friends and staff in the Department of Pharmaceutics for their friendship and support, especially to Whocely Victor De Castro for his suggestions during the validation of the analytical methods. I also w ould like to thank Pattara porn Vanachayangkul for her friendship. I would like to extend my thanks to the pr ogram assistants of the Department of Pharmaceutics Mr. James Ketcham, Mrs. Patric ia Khan, Ms. Michelle Griffin, Mrs. Andrea Tucker, and Mrs. Penny Canino for their technical support. I also would like to thank all my assistants: Carmen Michalski, Sandra Weiss, Eva Kremser, and Christine Haefele and the postdoc fellows, especially Dr. Vipul Kumar and Dr Jie Wang for their friendship and technical support. My personal thanks go to my mother and my father for their love, friendship, support, guidance and encouragement throughout my life.

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES................................................................................................................ .......11 ABSTRACT....................................................................................................................... ............13 CHAPTER 1 INTRODUCTION..................................................................................................................15 Artichoke ( Cynara scolymus L.).............................................................................................15 Pharmacological Actions.................................................................................................15 Constituents................................................................................................................... ..16 Dosage......................................................................................................................... ....17 Absorption and Metabolism of Caffeolyquinic Acids............................................................17 Absorption and Metabolism of Flavonoids............................................................................17 Biological Effects of Flavonoids............................................................................................19 Antioxidant Activity........................................................................................................19 Xanthine Oxidase Inhibitors............................................................................................20 Uric Acid, Hyperu ricemia, and Gout......................................................................................22 Enzyme Inhibition.............................................................................................................. ....23 Competitive Inhibition.....................................................................................................24 Uncompetitive Inhibitions...............................................................................................24 Mixed Inhibitions or Non Competitive Inhibitions.........................................................24 Pharmacokinetics............................................................................................................... .....24 Hypothesis and Objectives.....................................................................................................25 2 IDENTIFICATION AND QUANTIFICATI ON OF COMPOUNDS IN ARTICHOKE EXTRACT........................................................................................................................ ......40 Background..................................................................................................................... ........40 Specific Aim................................................................................................................... ........40 Materials and Methods.......................................................................................................... .40 Materials...................................................................................................................... ....40 Sample Preparation..........................................................................................................41 HPLC/DAD Analysis......................................................................................................41 Work Solutions and the Prepara tion of Calibration Standards........................................41 Quantification................................................................................................................. .42 Validation..................................................................................................................... ...42 Results........................................................................................................................ .............43 Linearity...................................................................................................................... ....43 Sensitivity.................................................................................................................... ....43

PAGE 6

6 Specificity.................................................................................................................... ....43 Precision and Accuracy...................................................................................................44 Stability...................................................................................................................... ......44 Quantification of Caffeoylquinic Acids (Chl orogenic Acid, Cynarin) and Luteolin Derivatives (Luteolin-7-Oglucoside, Luteolin-7-O-glucuronide) in Artichoke Leaf Extract..................................................................................................................44 Discussion and Conclusion.....................................................................................................44 3 EFFECT OF ARTICHOKE LEAF EXTRAC T, CAFFEIC ACID DERIVATIVES AND FLAVONOIDS ON XANTHINE OX IDASE INHIBITORY ACTIVITY............................54 Background..................................................................................................................... ........54 Specific Aim................................................................................................................... ........54 Materials and Methods.......................................................................................................... .54 Materials...................................................................................................................... ....54 Preparation of Working Solu tions and Test Solutions....................................................55 Assay Procedure for Xanthine Oxidase Inhibitions........................................................56 LineweaverBurk Plot....................................................................................................57 Results........................................................................................................................ .............57 Xanthine Oxidase Inhibitory Activity of Artichoke Extract...........................................57 Xanthine Oxidase Inhibitory Activity of Various Flavonoids and Compounds in Artichoke......................................................................................................................57 Inhibition Mechanism......................................................................................................58 Discussion and Conclusion.....................................................................................................58 4 EFFECTS OF ARTICHOKE LEAF EX TRACT AND VARIOUS FLAVONOIDS ON SERUM URIC ACID LEVELS IN OXONATE-INDUCED RATS.....................................67 Background..................................................................................................................... ........67 Specific Aim................................................................................................................... ........67 Materials and Methods.......................................................................................................... .67 Materials...................................................................................................................... ....67 Stock Solutions and Preparati on of Calibration Standards..............................................68 Animals and Experimental Protocols..............................................................................68 Animals....................................................................................................................68 Animal model of hyperuricemia in rats....................................................................69 Drug Administration:.......................................................................................................69 1. Oral administration...............................................................................................69 2. Intraperitoneal administration..............................................................................70 Uric Acid Assay..............................................................................................................70 Preparation of Rat Serum................................................................................................70 Statistical Analysis..........................................................................................................71 Validation..................................................................................................................... ...71 Results........................................................................................................................ .............72 Validation of Analytical Method to Measure Uric Acid in Rat Serum...........................72 Linearity...................................................................................................................72 Sensitivity.................................................................................................................72

PAGE 7

7 Specificity.................................................................................................................72 Precision, accuracy and recovery.............................................................................72 Stability....................................................................................................................72 Effect of Artichoke Extract and It s Compounds on Serum Urate Levels in Hyperuricemic Rats.....................................................................................................73 Oral administration of ar tichoke in acute treatment.................................................73 Oral administration of artic hoke in chronic treatment.............................................73 Oral administration of compounds in arti choke and various flavonoids in acute treatment...............................................................................................................73 Intraperitoneal administ ration of artichoke, compounds in artichoke and various flavonoids in acute treatment...................................................................74 Discussion and Conclusion.....................................................................................................74 5 THE EFFECT OF ARTICHOKE LEAF EXTRACT AND ITS COMPOUNDS ON ANTIOXIDANT ACTIVITY IN VITRO AND IN RATS....................................................92 Background..................................................................................................................... ........92 Specific Aims.................................................................................................................. ........93 Materials and Methods.......................................................................................................... .93 Materials...................................................................................................................... ....93 Animals........................................................................................................................ ....94 Acute treatment........................................................................................................94 Chronic treatment.....................................................................................................94 Assessment of Antioxidative Capacity in Vitro and Plasma Anti oxidant Status............95 Assessment of Uric Acid in Plasma................................................................................95 Assessment of Glutathione Pe roxidase (GPx) in Plasma................................................96 Statistical Analysis..........................................................................................................97 Results........................................................................................................................ .............97 Antioxidant Activity in Vitro ...........................................................................................97 Plasma Antioxidant Activity in Vivo ...............................................................................97 Acute treatment........................................................................................................97 Chronic treatment.....................................................................................................97 Plasma Urate Concentrations and Plasma Glutathione Peroxidase Activity after The Treatment with Artichoke Ex tract and Phenolic Compounds.....................................98 Discussion and Conclusion.....................................................................................................98 6 PHARMACOKINETICS OF LUTEOLIN AND ITS METABOLITES IN RATS.............108 Background..................................................................................................................... ......108 Specific Aims.................................................................................................................. ......108 Materials and Methods.........................................................................................................108 Materials...................................................................................................................... ..108 Stock, Work Solutions, and Prepar ation of Calibration Standards...............................109 Animals and Experimental Protocols............................................................................110 Animals..................................................................................................................110 Methods..................................................................................................................110 Analytical Methods.......................................................................................................111

PAGE 8

8 Data Analysis.................................................................................................................112 Statistical Analysis........................................................................................................114 Validation..................................................................................................................... .114 Results........................................................................................................................ ...........114 Validation of Analytical Method to Measure Luteolin in Rat Plasma..........................114 Linearity.................................................................................................................114 Sensitivity...............................................................................................................115 Specificity...............................................................................................................115 Precision, accuracy and recovery...........................................................................115 Stability..................................................................................................................115 Validation of Analytical Method to Measure Luteolin in Rat Urine.............................116 Linearity.................................................................................................................116 Sensitivity...............................................................................................................116 Specificity...............................................................................................................116 Precision, accuracy and recovery...........................................................................116 Stability..................................................................................................................116 Pharmacokinetic Study of Luteolin...............................................................................117 Non-compartmental analysis.........................................................................................117 Compartmental Analysis...............................................................................................118 Discussion and Conclusion...................................................................................................118 7 CONCLUSION.....................................................................................................................139 LIST OF REFERENCES.............................................................................................................141 BIOGRAPHICAL SKETCH.......................................................................................................151

PAGE 9

9 LIST OF TABLES Table page 1-1 Annual incidence of gouty arthritis according to the se rum urate concentration..............27 1-2 Drugs used in the management of gout..............................................................................28 2-1 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of chlorogeni c acid, cynarin, luteolin-7-O -glucoside and luteolin-7O-glucuronide.................................................................................................................. ..46 2-2 The stability test of chlorogenic acid, cynarin and luteolin-7 -O-glucoside after 24 hours on autosampler at 20oC............................................................................................47 2-3 The stability test of lu teolin-7-O-glucuroni de after 24 hours on autosampler at 20oC. Data represents the percentage remaining of all test compounds......................................48 2-4 Intra-day (n = 3) and inter-day (n = 9) assay parameters of caffeoylquinic acid (chlorogenic acid and cynarin) and luteolin derivatives (luteolin-7-O-glucoside and luteolin-7-O-glucuronide)..................................................................................................49 2-5 Amounts of caffeoylquinic acids and luteolin derivatives expressed as milligram per gram of dried extract..........................................................................................................50 3-1 Structures of various flavonoids........................................................................................61 3-2 Results of the % XO inhibition screening of artichoke extract.........................................62 3-3 The IC50 values ( M) of test samples on xant hine oxidase inhibition...............................63 3-4 Vmax and Km of flavonoids on xanthi ne oxidase inhibition................................................64 4-1 Concentrations of the standard solutions used for the calibration curves and quality controls (QCs) of uric acid.................................................................................................77 4-2 Intra-day (n = 3), inter-da y (n = 9), and recovery (n = 3) assay parameters of uric acid in rat serum.............................................................................................................. ...78 4-3 The stability test after 24 hours on autosampler at 20oC...................................................79 4-4 Hypouricemic effects of allopurinol, water extract of artichoke on plasma urate levels ( g/mL) in oxonate-pretreated ra ts in acute treatment............................................80 4-5 Hypouricemic effects of allopurinol and artichoke extr act on plasma urate levels ( g/mL) in oxonate-pretreated rats after chronic treatment...............................................81

PAGE 10

10 4-6 Hypouricemic effects of allopurinol, apigenin, eriodict yol, luteolin, luteolin-7-Oglucoside, naringenin, quercetin on plasma urate levels ( g/mL) in oxonatepretreated rats after oral administration.............................................................................82 4-7 Hypouricemic effects of allopurinol, apigenin, eriodict yol, luteolin, luteolin-7-Oglucoside, naringenin, quercetin on plasma urate levels ( g/mL) in oxonatepretreated rats af ter i.p injection........................................................................................83 5-1 ORAC values of artichoke extract...................................................................................101 5-2 Relative ORAC values of pure chemicals with antioxidant activity...............................102 5-3 ORAC values of plasma samples.....................................................................................103 5-4 ORAC values of plasma samples.....................................................................................104 5-5 Plasma urate concentrations in rats af ter administration of artichoke extract and phenolic compounds........................................................................................................105 5-6 Plasma glutathione peroxidase activity in rats after administration of artichoke extract and phenolic compounds......................................................................................106 6-1 Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in plasma................................................................................122 6-2 Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in urine...................................................................................123 6-3 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of luteolin in rat plasma.................................................................................................................. ...124 6-4 The stability test after 48 hours on autosampler at 18oC.................................................125 6-5 Intra-day (n = 3), inter-day (n = 9), and recovery (n = 3) assay parameters of luteolin in rat urine................................................................................................................... .....126 6-6 The stability test of luteolin in urine after 48 hours on autosampler at 18oC..................127 6-7 Pharmacokinetic parameters of luteolin afte r oral and iv administ ration of luteolin at dose 50 mg/kg..................................................................................................................128 6-8 Pharmacokinetic parameters of luteolin co njugates after oral and iv administration of luteolin at dose 50 mg/kg.................................................................................................129 6-9 Pharmacokinetic parameters of luteolin af ter oral and i.v. admini stration of luteolin 50 mg/kg....................................................................................................................... ...130 6-10 The excretory recovery for 24 h of luteolin and luteolin conjugates in urine after oral and i.v administration of lu teolin at dose 50 mg/kg.........................................................131

PAGE 11

11 LIST OF FIGURES Figure page 1-1 Structures of caffeoylquinic acids and flavonoids detect ed in artichoke ..........................29 1-2 Hypothetical metabolic path way of caffeoylquinic acids..................................................30 1-3 Proposed metabolic pathway of caffeic acid in isolated rat hepatocytes...........................31 1-4 Proposed recycling of flavonoids thr ough sequential metabolism and/or secretion involving intestinal microflo ra, intestine, and liver...........................................................32 1-5 The enzymatic process cata lyzed by xanthine oxidase......................................................33 1-6 Purine degradation pathway in animals.............................................................................34 1-7 The mechanism of uricase and uricase inhibitors..............................................................35 1-8 Enzyme inhibition.......................................................................................................... ....36 1-9 Competitive inhibition..................................................................................................... ..37 1-10 Uncompetitive inhibition.................................................................................................. .38 1-11 Mixed inhibition.......................................................................................................... .......39 2-1 Mean calibration curves of compounds in artichoke.........................................................51 2-2 HPLC separation and abso rbance-wavelength spectra of chlorogenic acid, cynarin, dihydrocaffeic acid, luteo lin-7-O-glucoside and lute olin-7-O-glucuronide......................52 2-3 Absorbance-wavelength spectras.......................................................................................53 3-1 Inhibition dose-response effects........................................................................................65 3-2 Lineweaver-Burk plots in the absence (control, ) and in the presence of luteolin (0.5 M, ), apigenin (0.5 M, ), kaempferol (0.5 M, ) and quecetin (0.5 M, ) with xanthine as the substrate.................................................................66 4-1 Mean calibration curves (n = 9) of uric in serum..............................................................84 4-2 HPLC chromatogram of uric acid in serum.......................................................................85 4-3 Acute effects of allopurinol artichoke extract on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate.....................................................................86 4-4 Chronic effects of allopu rinol, artichoke extratc on se rum urate levels in oxonatetreated rats................................................................................................................... .......87

PAGE 12

12 4-5 Effects of allopurinol and lu teolin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate...................................................................................88 4-6 Effects of allopurinol, apigenin, eriodict yol, luteolin-7-O-glucoside, naringenin, and quercetin on serum urate levels in rats pret reated with the uricase inhibitor potassium oxonate........................................................................................................................ .......89 4-7 Effects of allopurinol, apigenin, eriodi ctyol, luteolin-7-O-glucoside, naringenin, quercetin on serum urate levels in rats pret reated with the uricase inhibitor potassium oxonate........................................................................................................................ .......90 4-8 Effects of artichoke extr act, allopurinol, caffeic acid, chlorogenic acid, cynarin, luteolin, apigenin and quercetin on serum ur ate levels in rats pretreated with the uricase inhibitor potassium oxonate...................................................................................91 5-1 Structures of caffeic aci d derivatives and flavonoids......................................................107 6-1 Two-compartment models af ter intravenous injection....................................................132 6-2 Mean calibration curves (n = 9) of luteolin in plasma.....................................................133 6-3 The HPLC chromatogram of luteol in and naringenin (IS) in plasma..............................134 6-4 Mean calibration curves (n = 9) of luteolin in urine........................................................135 6-5 The HPLC chromatogram of luteol in and naringenin (IS) in urine.................................136 6-6 Plasma concentration-time curves...................................................................................137 6-7 Fitted luteolin concentra tions after i.v. injection.............................................................138

PAGE 13

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 XANTHINE OXIDASE INHIBI TION AND ANTIOXIDANT ACTI VITY OF ARTICHOKE LEAF EXTRACT ( Cynara scolymus L.) AND ITS COMPOUNDS By Sasiporn Sarawek August 2007 Chair: Veronika Butterweck Cochair: Hartmut Derendorf Major: Pharmaceutical Sciences Gout is a disease characterized by elevated levels of uric acid in body fluids. This hyperuricemia results in th e deposition of urate crysta ls in tissue, especially joints. The uric acid deposition initiates an inflamma tion process involving the rele ase of reactive oxygen species. The common treatments of gout are the use of anti -inflammatory agents to relieve the symptoms of the disease and xanthine oxidase (XO) inhibitors to block the s ynthesis of uric acid. The most common xanthine oxidase inhibito r is allopurinol. However, its use is limited by unwanted side effects such as hypersensitivity problems. Therefore, alternatives are required. Artichoke leaves (Cynara scolymus L .) have been used traditionally by the Eclectic physicians as a diuretic and depurative for th e treatment of gout. The major compounds in artichoke leaves are phenolic compounds such as caffeoylquinic acids and flavonoids. These phenolic compounds have shown xanthine oxidase inhibitory activ ity and antioxidant activity in vitro and in vivo Therefore, the goal of the present st udy was to examine the xanthine oxidase inhibitory activity and antioxidant activity of the artichoke extract, and its main compounds in vitro and in vivo

PAGE 14

14 The in vitro study showed that the extract as well as caffeoylquinic acids showed only a weak XO inhibition, whereas flavonoids (flavone a nd flavonols) had a highl y inhibitory effect on XO. Luteolin had the highest XO inhibition eff ect. This significant inhibition of XO by the flavonoids in vitro suggested that they may supp ress the production of uric acid in vivo However, the in vivo study showed that oral administration of the artichoke extract, caffeoylquinic acids, and flavonoids could not decrease the serum ur ate levels in oxonate-treated rats. The antioxidant activities of the artichoke extract and its phenolic compounds were determined using the oxygen radical absorbance capacity assay (ORAC). The results showed that the artichoke extract and its compounds elicited an antioxidant activity in vitro however, the compounds again showed no antioxidant activity in vivo It was speculated that this lack of effect in vivo from both studies might be due to the absorption, the high first pass effect through intestin e and liver, the excretion into urine and bile and the degradation in large intestine. Th erefore, the pharmacokinetic of a compound in artichoke was performed in order to explain the in vivo activity. Pharmacokinetic study of luteolin, th e compound which showed the highest XO inhibition in vitro showed that after oral administration of luteolin, luteolin rapidly absorbed and metabolized in plasma. Additionally, plasma-concentration-time curves of luteolin metabolites revealed secondary peaks. The bioavailability of luteolin was low and th e urinary excretion of luteolin and its conjugates did not dominate. This study could expl ain the lack of XO inhibitory activity and antioxidant activity in vivo Therefore, it can be concl uded that artichoke might be not a useful alterna tive treatment of gout.

PAGE 15

15 CHAPTER 1 INTRODUCTION Artichoke ( Cynara scolymus L.) Artichoke or globe Artichoke (Cynara scolymus L .) a member of the Compositae (daisy) family, is native to the Mediteranean area. The leaves are the commonly used part. Pharmacological Actions Traditionally artichoke leaves have been used by the Eclectic physicia ns as a diuretic and depurative, for the treatment of rheumatism, gout, jaundice and especially for dropsies. For the modern use, the leaf of artic hoke is reported to process choler etic, hypocholestero laemic [1, 2], hypolipidaemic [3], hepatoprotective [4], anticar cinogenic [5] and antioxidative [6-9]. Diuretic effect of artichoke helps the elim ination of water and the consequent toxin and specially the uric acid. Hypolipidaemic, hypocholesterolaemic and chol eretic activities are well documented for artichoke leaf extracts and part icularly for the constituent cynari n [10]. Artichoke leaves not only increases choleresis and, theref ore, cholesterol elimination, but also has been shown to inhibit cholesterol biosynthesis. Clinical trials investigating the use of globe artichoke and cynarin in the treatment of hyperlipidae mia report positive results [10]. However, studies in animals and humans by Saenz et al. [11] have suggested that these effects may be due to the monocaffeoylquinic acids present in artic hoke extract (eg. chlorogenic acid) [11]. In vitro studies on cultured hepatocytes suggested that artichoke extract inhib its the incorpor ation of 14Clabelled acetate into the non-sa ponifiable lipid fraction and thus reduce cholesterol biosynthesis [12, 13]. Other studies suggested indirect inhibitory effects ex erted at the level of HMGCoA reductase, a key enzyme in c holesterol biosynthesis [13-15].

PAGE 16

16 Antioxidant and hepatoprotective activity of artichoke leaves have been studied. In vitro a luteolin-rich artichoke leaf extract (flavonoi d content around 0.4% w/w), the pure aglycone luteolin, and luteolin-7-O-glucoside demonstrat ed a concentration-dependent reduction in low density lipoprotein (LDL) oxidation [9]. The eff ects of artichoke extract a nd its constituents have also been investigated for activity against oxida tive stress in studies using human leucocytes. The extract demonstrated a concentration-dependent inhibition of oxidative stress induced by several agents, such as hydrogen peroxide, that genera te reactive oxygen speci es. The constituents cynarin, caffeic acid, chlorogenic acid and lute olin also showed concentration-dependent oxidative stress inhibitory activ ity [16]. In addition, artichoke extract has marked protective properties against oxidative st ess induced by inflammatory me diators and ox-LDL in cultured endothelial cells and monocytes [7]. In vivo the administration of an ed ible artichoke in rats has shown that artichoke extract incr eased the level of glutathione pe roxidase activity in erythrocyte and decreased the level of 2-Aminoadipic semi aldehyde (a protein oxi dation biomarker) [8]. Hepatoprotective and hepatoregenerating activ ities have been documented for cynarin in vitro [4] and in rats [10, 17]. Artichoke extract has been reported to alle viated symptoms and improving the diseasespecific quality of life in patient s with functional dyspepsia [18] and concomitant dyspepsia [19]. Constituents The major chemical components of artichoke le aves include up to 2% phenolic acids with mono-and dicaffeoylquinic acids, primarily chlor ogenic acid, cynarin, and caffeic acid. Also up to 0.1-1% flavonoids (Figure 1-1) [20-24].

PAGE 17

17 Dosage The German Commission E recommends an av erage daily dose of 6 g drug, or an equivalent dose of extract (based on the herb-toextract ratio) or other pr eparations, for dyspeptic problems. A recommended dosage regimen for liquid extract (1: 2) is 3-8 ml daily. Dosage used in clinical trials of globe arti choke leaf extract have assessed the effects of dosages of up to 1.92 g daily in divided doses fo r up to six months [25]. Absorption and Metabolism of Caffeolyquinic Acids The mechanism and site of absorption of caffe oylquinic acids is stil l unclear. There is no published evidence for enzymatic hydrolysis of ch logenic acid by intestin e, liver or plasma extracts [26, 27]. Moreover, chloro genic acid has been reported to be stable in the digestive or intestinal juice [28, 29]. However, Wittemer et al. [30] sugge sted absorption and de-esterification of caffeoylquinic acids may occur somewhere in the upper gut. After the re lease of caffeic acid (CA) from caffeoylquinic acids, CA may be conjuga ted with glucuronic acid in the enterocytes [30]. After entering the systemic circulation, CA conjugates we re most likely methylated by catechol-O-methyltransferase [31] during the firs t liver passage to met hylation products of ferulic acid (FA) and isoferulic acid (IFA).They also suggested that CA may be metabolized by the colonic microflora to dihydrocaffeic acid ( DHCA) prior to absorption. In enterocyte, DHCA was metabolited to dihydroferuli c acid (DHFA) using catechol-O -methyltransferase, and then FA was formed by the dehydrogenation of DHFA [ 30, 31]. The hypothetical metabolic pathways of caffeoylquinic acids and caffeic acid are shown in Figure 1-2, and Figure 1-3. Absorption and Metabolism of Flavonoids Most of flavonoids presented in plants are a ttached to sugar moieties thus tending to be water-soluble, although, occasionally, they are found as aglycones. Absorption of flavonoid glycosides was considered negligible. Only fla vonoid aglycones were be able to pass the gut

PAGE 18

18 wall, and no enzymes that can split these -glycosidic bonds are secr eted into the gut or presented in the intestinal wall [32]. Hydrolysis occurs in the colon by intestinal microflora, which could be further metabolized by intestinal microflora to various single-ring aromatic compounds [33, 34]. Hydrolytic enzy mes of intestinal microflora could convert certain flavonoid glycosides to their corresponding aglycones [33, 35]. Recently, it has been reported that enzymes that are able to hydrolyse fla vonoid glycosides are located in the cells (cytosolic betaglucosidase, CBG) and on the apical membrane (lactase-phlo rizin hydrolase, LPH) [36]. Therefore, flavonoid glycosides may be hydrolys ed by LPH and then the aglycone may diffuse passively into the cell [37]. Alte rnatively, flavonoid glycosides may be enter the cell by sodium dependent glucose transporter (SGTL1) [38] and then be cl eaved inside the cell by CBG. Absorbed flavonoids could undergo phase I (e.g., oxidation such as hydroxylation) and phase II (e.g., conjugation such as glucur onidation) metabolisms in human intestine and liver. Phase I metabolisms commonly attach a hydroxyl group to the molecule or break down a molecule so that the compound can be further processed by the body. Phase II metabolisms can occur after phase I metabolisms or simultaneously as phase I. Normally, phase II metabolisms convert compounds and their phase I metabo lites into hydrophilic and excr etable conjugates which could be eliminated by the urine or via the bile. In case of flavonoi ds, conjugated metabolites are finally excreted into the intestinal lumen and eliminated or be hydrolysed by microbial hydrolases (e.g., glucuronidases and sulfatase) at the intestinal lumen to aglycones, and then transported into systemic circul ation. (Figure 1-2) The low bioava ilability of flavonoids may be explained by duoenteric and ente rohepatic recirculation [39].

PAGE 19

19 Biological Effects of Flavonoids Flavonoids have been shown to exert protective effects against many diseases, in particular cardiovascular diseases and cancer The health benefit of flavonoi ds is usually linked to two properties: (1) antioxidant activity and (2) inhibiti on of certain enzymes such as xanthine oxidase [40, 41]. Antioxidant Activity Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide radical anion (O2 -), hydroxyl radical (OH), alkylperoxyl radical (ROO), nitric oxide (NO), singlet oxygen (1O2) and hypochlorous acid (HOCl) react with biological molecules causing cell and tissue injury. The ROS are consider ed to contribute to a wide va riety of degenerative processes and diseases such as atherosclerosis, Parkinson s disease, Alzheimers dementia and reperfusion injury of brain or heart [42]. Many studies have suggested that flavonoi ds exhibit biological activities, including antiallergenic, antiviral, anti-inflammatory, vasodilating actions. These pharmacological effects are linked to the antioxi dant properties of flavonoids. Flavonoids can express these properties by: (1 ) suppressing ROS formation by inhibiting some enzymes or chelating trace elements involve d in free radical production, (2) s cavenging radical species and more specially the ROS, or (3) up-regulati ng or protecting antioxidant defense [40]. Flavonoids can inhibit enzymes which are re sponsible for superoxi de anion production such as xanthine oxidase. Most of flavonoids can chelate trace metals, wh ich play an important role in oxygen metabolism, and therefore inhibit the initiation of the li poxygenase reaction [43]. The possible metal-complexing site s within flavonoids are loca ted between the C3 hydroxyl and the carbonyl, the C5 hydroxyl a nd the carbonyl and be tween the ortho-hydroxyls on the B-ring [40]. The radical scavenging activity of flavonoids depend on the structure and the substituents of the heterocyclic and B-ring. The major determ inants for radical-scaven ging capacity are: (1)

PAGE 20

20 the otho-dihydroxy structur e on the B-ring, which has the best electron-donating properties, (2) the 2, 3-double bond in conjugation w ith a 4-oxofunction in the C-ri ng is responsible for electron delocalization from the B-ring, and (3) the 3-and 5-hydroxyl group with a 4-oxofunction in the A and C-ring for maximum radical scavenging potent ial [40]. Some flavonoids, such as qurcetin, myricetin, and fisetin, were shown to alleviat e oxidative stress by inducing glutathione Stransferase (GST), an enzyme used to protect ce lls against free-radical da mage [44]. Studies have indicated that flavonoid aglycone s, including quercetin, luteolin, myricetin, and kaempferol have greater antioxidant capac ity than their glycosides such as que rcetin-3-glucoside [45]. Noroozi et al. [45] reported that, at equimolar concentratio n, most flavonoids show ed greater antioxidant capacity than vitamin C. Currently, the relevance of in vitro studies to the in vivo situation is unclear. Terao et al. [46] found that oral administra tion of (-) epicatechin and quercetin enhanced the antioxidant capacity of rat plasma, although both flavonoids a ccumulated mainly as glucuronide and sulfate conjugates in blood plasma. Morand et al. [47] had reported that the conjugate metabolites of quercetin could inhibit the oxida tion of LDL catalyzed with Cu+2.[47] Janisch et al.[48] found that flavonoid intestinal and hepatic metabolism had an abilit y to inhibit LDL oxidation. These finding suggests that conjugated metabolites of fl avonoids may play a role in the antioxidant defenses of blood plasma. In human, Arai et al. [49] found total inta ke of flavonoids among women to be inversely correlated with plasma total cholesterol and low density lipoprotein concentrations, after adjustment for age, body mass index, and total energy intake. Further in vivo experiments are needed to explore. Xanthine Oxidase Inhibitors Xanthine oxidase has a role in the generation of ROS in various pathologies such as viral infection [50], inflammation [51], brain tumors [52] or the pro cess of ischemia/reperfusion [53,

PAGE 21

21 54] has been studied. Xanthine oxidase belongs to the molybdenum-protein family containing one molybdenum, one of the flavin adenine dinu cleotide (FAD) and two iron-sulfur (2Fe 2S) centers of the ferredoxine type in each of its two independent subunits. Xanthine oxidase is a cytosolic enzyme found in many species such as bacteria, higher plan ts, invertebrates and vertebrates [55]. It is present in the liver, in testine, kidney, lungs, myocardium, brain, plasma, and erythrocytes, and other tissu es of several mammalian specie s including human [56]. In all mammals, the liver and intestine have the highes t xanthine oxidase activ ity [55]. This enzyme catalyzes the conversion of both hypoxanthine to xanthine and xa nthine to uric acid while reducing O2 to O2 and H2O2 according to Figure 1-5 [57]. The enzyme contains two separated substratebinding sites. Xanthine oxidase inhibitors can act either at the purine binding site such as allopurinol [58, 59] or at the FAD cofactor site such as benzimidazole [60]. Allopur inol is a potent inhi bitor of xanthine oxi dase which has been widely used to treat gout and hype ruricemia [61, 62]. However, severe toxicity of allopurinol such as vasculitis, rash, eosinophilia, hepatitis has been reported [63]. Currently, no clinically effective xanthine oxidase inhib itor for the treatment of hyperuricemia has been developed since allopurinol. Therefore, new inhi bitor devoid of undesired side e ffects has been investigated. Many studies of natural polyphenols, especially fl avonoids, in the form of plants or purified extracts show that they could be used as xa nthine oxidase inhibitors [64-66]. The essential structural characteristics for the inhibition of xanthine oxidase ar e (1) the presence of the benzo-pyrone structure (2) the presen ce of free hydroxyl groups at positions 7, 3 and /or 5 in the flavonoid structure [40] and (3) an -unsaturated carbonyl group that helps electronic delocalization of phe nyl ring B [40, 56].

PAGE 22

22 Different types of inhibition are found concerning xanthine and flavonoids as substrates: competitive, non competitive and mixed type inhibition. Different modes of inhibition were demonstrated at steady state measurements using Lineweaver-Burk plots. In the mixed inhibition, the inhibitor can bind to the free enzyme and to the enzyme-substrate complex [40]. Uric Acid, Hyperuricemia, and Gout Uric acid is produced by the degradation of purine co mpounds either from exogenous (dietary) or endogenous origin (Figure 1-6). Most species, except humans, some apes and the dalmatian dogs have rather low blood levels of uric acid because of the presence of th e uric acid catabolizing enzyme uricase in the plasma and liver [67]. Uricase tr ansforms uric acid to allantoin, which is water soluble and can be excreted. Thus, in rat experiment, we have to use an uricase inhibitor such as potassium oxonate to increase endogenously synt hesized uric acid (Figure.1-7). At physiological pH almost all uric acid is i onized to urate since the pKa of uric acid is around 5.4. Urate has limited solubility in water. Therefore, the excess production of uric acid can lead to the deposition of ur ate crystals in various locals, particulay in the joints, the connective tissues, and the kidneys [68]. Hyperuri cemia is generally the cause for gout which is characterized by a serum uric acid level of above 7.5 mg per 100 mL for males and 6.6 mg per 100 mL for females [69]. Gout occurs when urate monohydrate crystals deposit in the joint sp ace between two bones or in both. These depositions lead to inflammatory arthritis, which causes swelling, redness, heat, pain, and stiffness in the joints. The infla mmatory response involves local infiltration of granulocytes, which phagocyte th e urate crystals. This proces s generates oxygen metabolites, which damage tissue, and results in the rel ease of lysosomal enzymes that inducing an inflammatory response. Moreover, lactate produc tion is high in synovial tissues and in the

PAGE 23

23 leukocytes associated with the inflammatory proce ss. The high level of la ctate leads to a local decrease in pH that fosters furt her deposition of uric acid. In fa ct, the major risk factor for the development of gout is sustained asymptomatic hyperuricemia (Table 1-1) [70].The optimal diagnosis of gout is the demonstr ating urate crystals in synovia l fluid or a tophus (a nodular collection of urate crystals in soft tissue) [70, 71]. The commonly report of gout is 6 per 1000 population in men and 1 per 1000 population for women [71]. The incidence of gout has b een found to be increasing [72, 73]. With the Rochester Epidemiology Project computerized medical record system, the incidence rate increase more than twofold from 19771978 to 1986-1987 in Rochester, MN [73]. The goal of antihyperuricemic therapy is to reduce serum uric acid level below the threshold required for supersatura tion of extracellular fluid, to pr event or reverse tissue damage resulting from uric acid depositi on, and to decrease the inciden ce of recurrent attacks of gout arthritis [69, 74]. Drugs used to reduce uric acid leve ls can be either uricosuric drugs or xanthine oxidase inhibitors [74]. All the synthetic drugs used in the treatment of gout (Table 1-2) have some side effects, therefore an alternative are required. Enzyme Inhibition The basic equation of enzyme kinetics is Michaelis-Menten equation (V = Vmax [S]/ Km + [S]). This equation has the same form as the equation for a rectangular hyperbola; the reaction rate (V) versus substrate concen tration [S] produces a hyperbolic ra te plot (Figure 1-8). To avoid dealing with curvilinea r plots of enzyme catalyzed reacti ons, the Lineweaver-Burk plot was introduced (Figure 1-8).The equation of Lineweaver-Burk is [1/V] = [Km (1)/ Vmax[S] + (1)/Vmax] [75].

PAGE 24

24 Enzyme inhibitors are substances that reduce an enzyme activity and have similar structure to their enzymes substrate but e ither does not react or react very slowly compared to substrate. The mechanisms of inhibiti on are described as follow. Competitive Inhibition A substance that competes directly with a norm al substrate for an enzymatic binding site is known as a competitive inhibitor. These inhibito rs usually resemble the substrate and act by reducing the concentration of free enzyme availa ble for substrate binding. The general model for competitive inhibition and the Lineweaver-Bur k plot are showed in Figure 1-9 [75]. Uncompetitive Inhibitions The inhibitor binds directly to the enzymesubstrate complex but not to the free enzyme as shown in Figure 1-10. Mixed Inhibitions or Non Competitive Inhibitions The inhibitors bind to both the enzyme and enzyme-substrat e complex bind inhibitor as shown in Figure 1-11. Pharmacokinetics Pharmacokinetics (PK) is defined as the st udy of the time course of drug absorption, distribution, metabolism and excr etion. Absorption describes th e process of drug molecules moving from the site of administration to sy stemic circulation. Dist ribution describes the movement of drug molecules from systemic circ ulation to extravascular sites. Metabolism describes the enzymatic breakdown of drugs. It is frequently a primary defense mechanism used by the body to avoid exposure to xenobiotics. Drugs molecules are converted to more hydrophilic metabolites and excreted from the body. Me tabolites can be inactive, active or toxic. Therefore, understanding th e pathway where a compound is metabolized and PK of its

PAGE 25

25 metabolites is essential. Finally, excretion descri bes passive or active transport of drug molecules into urine or bile [76]. Pharmacokinetics studies rely on the meas urement of the active drugs and/or its metabolites in biological fluid such as blood, plasma or urine. From this information, concentration-time curves may be constructed and pharmacokinetic parameters such as area under the curve (AUC), maximum concentra tion (Cmax), clearance (Cl), volume of distribution(Vd) and elimination half-life ( t 1/2) may be calculated [77]. Pharmacokinetics is also applied to therap eutic drug monitoring (TDM) for very potent drugs such as those with a narrow therapeutic rang e, in order to optimize efficacy and to prevent any adverse toxi cities [78]. Hypothesis and Objectives Gout is a common disease with a worldwide distribution and continues to be a health problem. It is often associated with elevated serum levels of uric acid. The most common symptom in gout is painful arthritis joint inflammation, caused by deposition of insoluble crystals of sodium urate. Nowadays, it seems to be accepted that the key factor to control this disease is the prevention and th e treatment. The treatment of gout includes the use of antiinflammatory agents such as non-steroidal anti -inflammatory drugs (NSAIDs) for symptomatic relief and xanthine oxidase i nhibitors to block th e endogenous production of uric acid. However, NSAIDs produce side effects such as naturopathy, nitrog en retention, and, hyperkalemia. Allopurinol, the most common xanthine oxidase inhi bitor, also has unwanted side effects such as hypersensitivity problems. Therefore, al ternative treatments are required. The leaves of artichoke have been used traditionally by the Eclectic physicians as a diuretic and depurative, for treatments of rheumatism, gout, ja undice and especially for dropsies. The major compounds of artichoke are phenolic compounds such as caffeoylquinic acids and

PAGE 26

26 flavonoids. The phenolic compounds have shown xa nthine oxidase inhibition and antioxidant activity in vitro and in vivo Therefore, artichoke leaves c ontaining polyphenolic compounds may show xanthine oxidase inhibito ry activity and antioxidant ac tivity. In the present study the xanthine oxidase inhibitory activ ity and antioxidant activity of artichoke extract, and its main constituents were investigated in vitro and in vivo Furthermore, the pharmacokinetic of an activ e compound in artichoke extract was studied in male Sprague-Dawley rats in order to assess the in vivo efficacy and obtain more information about absorption and disposition. The concentra tion of a single compound and its metabolites will be detected in plasma and urine and pharmacokinetic parameters will be calculated. Therefore, to test the hypothesis of this study the following specific aims were purposed: Specific aim#1: Phytochemical investigation of co mpounds in artichoke extract. Specific aim#2: Determine whether artichoke extract a nd its compounds show the inhibition of xanthine oxidase in vitro Specific aim#3: Investigate whether artichoke extract and its compounds can decrease uric acid in rat serum. Specific aim#4: Determine whether artichoke extract and its compounds show antioxidant activity in vitro and in vivo. Specific aim#5: Pharmacokinetic analysis of an active compound in artichoke extract.

PAGE 27

27 Table 1-1. Annual incidence of gouty arthritis acco rding to the serum urate concentration [70]. Serum Urate Concentration (mg/dl )Annual Incidence of Gout (%) <7.00.1-0.5 7.0 8.90.5-1.2 9.0 4.9-5.7

PAGE 28

28 Table 1-2. Drugs used in the management of gout [79, 80]. Drug Comment To treat acute gouty arthritis Colchicine Inhibits crystal phagocytosis; no eff ect on urate metabolism; increased toxicity in patients who have renal or hepatic dysfunction or are receiving concomitant therapy with P-450 enzyme inhibitors such as cime tidine, erythromycin, and tolbutamide [79]; current treatment is an intravenous dose of 2 mg, diluted in 10 to 20 mL of 0.9% sodium chloride solution; a total dose of 4 mg should not be exceeded. To avoid cumulative toxicity, treatment with colchicines should not be repeated within 7 days [80]. NSAIDs Effective in relieving pain and reducing inflammation in patients with acute gout but use limited by side effects (naturopathy, nitrogen retention, reduced creatinine clearance, hyperkalemia, abnormal liver-func tion values, and headache); greater risk of side effects in patients with renal dysfunction [79, 80]. Corticosteroids Effective either by intraarticular (single joint) or systemic route (intramuscular, intravenous, or oral); potential for rebound inflammation and side effects; administered only when NSAIDs and colchicines have been ineffective or are contraindicated [79, 80]. To prevent acute attacks Colchicine Effective in an oral dose (0.5-1.8 mg per day) adjusted so as not to cause diarrhea [80]. NSAIDs Useful if colchicine al one is insufficient and acute att acks recur frequently; usual dose is 150 to 300 mg of indomethacin per day or its equivalent [79]. To lower serum urate concentrations Probenecid Increases urate excretion by inhibits ur ate reabsorption at renal tubule; interferes with excretion of many drugs; serious toxic effects rare, although nausea and rash reported in up to 10 % of patients [79]; effective in an oral dose of 250 mg twice daily for 1 week, following with 500 mg twice daily for chronic treatment [80]. Allopurinol Inhibits xanthine oxidase; common si de effect are hypersen sitivity reactions [80]

PAGE 29

29 OH O H O OH OR4OR3OR2HOOC OR1 Caffeic acid Quinic acid Chlorogenic acid: R1=H, R2=H, R3=H, R4=caffeoyl 3,5-di-O-caffeoylquinic acid (Cynarin): R1=caffeoyl, R2=caffeoyl, R3=H, R4=H 3,5-di-O-caffeoylquinic acid: R1=H, R2=caffeoyl, R3=H, R4=caffeoyl 4,5-di-caffeoylquinic acid: R1=H, R2=H, R3=caffeoyl, R4=caffeoy O O OH R1O R2OH O O OH R1O OH luteolin-7-O-glucoside: R1=glc, R2=OH narirutin: R1=rutinose luteolin-7-O-rutinoside: R1=rut, R2=OH naringenin-7-O-glucoside: R1=glucose apigenin-7-O-glucoside: R1=glc, R2=H apigenin-7-O-rutinoside: R1=rut, R2=H Figure 1-1. Structures of caffe oylquinic acids and flavonoids detected in artichoke [22].

PAGE 30

30 OH OH O H O OH OMe O H O OMe OH O H O OMe OH O H O OH OH O H O OR OR HOOCOR ORCCA IFA, IFA-Conj. CA, CA-Conj. DHFA, DHFA-Conj. DHCA DHCA-Conj. L I V E RC O L O NS M A L L I N TE S T I N EC O L O N Figure 1-2. Hypothetical metabolic pa thway of caffeoylquinic acids [30].

PAGE 31

31 O O COOH OH OH COOH OH OH COOH O O COOH COOH OH OCH3 OH OCH3 COOH GS COOH OH OH GS COOH OH OH CYP 2E1 O2 or O2Acyl Co A dehydrogenase (ATP, CoA) Hydrogenase ? CYP 2E1 o-quinoneGS-CA conjugate Hydrogenase? Acyl Co A dehydrogenase (ATP, CoA) GSHC O M TC Y P 1 A 1 / 2C Y P 1 A 1 / 2C O M TGSH O 2 or O 2 FA DHFA Figure 1-3. Proposed metabolic pathway of caffeic acid in isolated rat hepatocytes [31].

PAGE 32

32 Figure 1-4. Proposed recycling of flavonoids th rough sequential metabolism and/or secretion involving intestinal microflora intestine, and liver. In this scheme, flavonoids are assumed to be given orally. This recyc ling scheme involves dual loops: one is the classical enterohepatic recycling and the other is enteric recycling, where phase II metabolites formed and excreted by the small intestine could be reconverted to their aglycones again in the large intestine by th e bacteria and reenter the blood via the colon. In this figure, SGLT1 refers to a glucose transporter and MRP refers to multidrug resistant related protein. SGLT1 could participate in the absorptive transport of glycosides [81], whereas MRP c ould act as a gatekeeper that prevents the absorption of glycosides [39, 81].

PAGE 33

33 N N H N N H O N H N H N N H O O N H N H N H N H O O OH Hypoxanthine Xanthine Uric acid Figure 1-5. The enzymatic process ca talyzed by xanthine oxidase [57]. Xanthine oxidase Xanthine oxidase O2, H2O O2, H2O O2 -, H2O2 O2 -, H2O2

PAGE 34

34 Ficture 1-6. Purine degradati on pathway in animals [67]. IMP Inosine AMP Adenosine deaminase AMP deaminase GMP Guanosine nucleotidases Purine nucleoside phosphorylase (PNP) NH4 +N N H N N H O Xanthine oxidase Hypoxanthine O2, H2O H2O2 H2O2 O2, H2O xanthine oxidase Adenosine Guanine NH4 N H N H N N H O O N H N H N N H O O ON N H N N H O N H2 N H4 + xanthine urate

PAGE 35

35 N H N H N H N H O O O O NH NH2N H N H O O Figure 1-7. The mechanism of uricas e and uricase inhibitors [67]. Uricase O2 CO2 Uric acid Uricase Inhibitors Allantoin

PAGE 36

36 A B Figure 1-8. Enzyme inhibition. A) Michaelis-Menten plot. B) Lineweaver-bur k plot. V is defines as a intial velocity, [S] is the substrate concentration, Vmax is a maximum velocity and Km is a substrate concentration at of Tmax [75].

PAGE 37

37 A B Figure 1-9. Competitive inhibition. A) The model for competitive inhibition. B) LineweaverBurk plot of the competitively inhibited Michaelis-Menten enzyme. E is defined as enzyme, S is substrate, I is inhibitor; EI is enzyme-inhibitor complex and P is product. Note that Vmax, as defined as the maximum velocity of a reaction, is unchanged; Km, as defined by [S] required for ma ximal activity, is increase [75].

PAGE 38

38 A B Figure 1-10. Uncompetitive inhibition. A) The model for uncompetitive inhibition. B) Lineweaver-Burk plot of a single Michael is Menten enzyme in the presence of uncompetitive inhibitor. Note that Vmax is decreased; Km, as defined by [S] required for maximal activity, is decreased [75].

PAGE 39

39 A B Figure 1-11. Mixed inhibition. A) The model for mi xed inhibition. B) Lineweaver-burk plot of a simple Michaelis Menton enzyme in the pres ence of a mixed inhibitor. Note that Vmax is decreased; Km appears unaltered [75].

PAGE 40

40 CHAPTER 2 IDENTIFICATION AND QUANTIFICATI ON OF COMPOUNDS IN ARTICHOKE EXTRACT Background The variation of the conten t of mono-and dicaffeoylquinic acids and flavonoids in artichoke extracts has been repor ted [23, 82]. For example, the content of luteolin-7-O-glucoside and 1, 3-O-dicaffeoylquinic acid were reported to vary from 1002 to 1616 mg/kg of dried extracts and from 1292 to 30985 mg/kg of dried ex tracts, respectively [23] This deviation of phenolic compounds might affect the pharmacological activitie s of artichoke extracts. Specific Aim The objective of this study was to identify and quantify marker compounds in artichoke extract. Materials and Methods Materials Water extract of artichoke leaf (Cynara scolymus L .) was obtained from a German extract manufacturing company (Finzelb erg, Andernach, Germany). Dihydr ocaffeic acid (90-95%) and luteolin-7-O-glucoside (>90%) were purchas ed from Indofine Chemical Company, Inc. (Somerville, NJ, USA). Chlorogenic acid ( 95%) was purchased Sigma Chemical Company (St. Louis, MO, USA). Cynarin was purchased from Carl Roth GmbH+Co. (Germany). Acetonitrile (CH3CN) and trifluoroacetic acid (TFA ) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Luteolin-7-O-glucuronide used in this study was a kind gift from Prof. Dr. A. Nahrstedt, Institute of Pharmaceutical Biology and Phytoche mistry, University of Mnster, Germany. All aqueous solutions were prepared with deioni zed water obtained from a NANOPure system from Barnstead (Dubuque, IA, USA).

PAGE 41

41 Sample Preparation 500 mg of powdered extract of Cynara scolymus L was dissolved in 20.0 mL of MeOH/H2O (3:7) at 25 C for 5 min. Th e solutions were filtered (0.45 m) and were directly analyzed by HPLC/DAD. HPLC/DAD Analysis Samples were analyzed using a reverse-phase partition mode of HPLC with diode array detector. A Shimadzu VP series HPLC syst em (Kyoto, Japan) equipped with an SPD-M10Avp diode array detector was used for this wo rk. The column used was a 2504.0 mm i.d.(5 m ) Lichrospher 100 RP-18e (Merck KgaA, Germa ny).The column temperature was kept at 25oC. The eluents were (A) 0.3% TFA and (B) CH3CN. The following solvent gradient was applied: 5% B (5 min), 5-20% B (50 min), 20-5%B (15 min), total run time was 70 min. The injection volume for all samples was 10 L. Flow elution was 1 mLmin-1. Chromatograms were acquired at 330 nm for the caffeoylquinic acid and 350 nm for the luteolin derivatives. UV-Vis spectra were recorded in the range 200-400 nm Work Solutions and the Preparation of Calibration Standards Chlorogenic acid, cynarin, and lute olin-7-O-glucoside work solution s (400 g/mL) : The amount of 10.0 mg of chlorogenic acid, cynarin and luteolin-7-O-glucosi de were accurately weighed, and transferred to a 25.0 mL volumetric flask. The standards were then dissolved in and brought to volume with methanol. Luteolin-7-O-glucuronide work solution (500 g/mL) : The amount of 1.0 mg of luteolin-7-O-glucuronide was weighted, and transferred to a 2.0 mL volumetric flask. The standard was then dissolved in methanol to obtain a final concentration of 500 g/mL. The volume was completed with same solvent, and the final solution mixed thoroughly.

PAGE 42

42 Standard solutions for chlorogenic acid cynarin, and luteolin-7-O-glucoside : From the chlorogenic acid, cynarin, and luteolin-7-O-g lucoside work solutions, five different concentrations of standard solutions of chlor ogenic acid, cynarin, and luteolin-7-O-glucoside and three quality controls (QC) were prepared in methanol according (Table 2-1). All solutions were filtered through a 0.45 m PVDF membrane filter (Millipore Corp.) before analysis. Standard solutions for luteolin-7-O-glucuronide : From the luteolin-7-O-glucuronide work solution, six different concen trations of standard solutions of -7-O-g lucuronide and three quality controls (QC) were prepared according to Table 2-1. The final volume was filled up with methanol in 2.0 mL volumetric flask. A ll solutions were filtered through a 0.45 m PVDF membrane filter (Millipore Corp.) before analysis. Quantification Calibration was carried out by an external standardization method. 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 method was validated over the range of c oncentration of the ta rget compounds present in the artichoke extracts. The validation parameters of linearity, sensitivity, specificity, precision, accuracy and stability were determined. The linearity of the calibration curves was de termined by least-squares linear regression method and expressed in terms of coefficient of determination (r2). The intraand inter-day

PAGE 43

43 precision and accuracy were measured by triplicate analyses of three different concentration levels (low, medium and high) of quality cont rol standards on the same day and on different days. The precision was based on the calculation coefficient of variation (CV %), and the accuracy was defined as the percent difference be tween the theoretical and measured values. The limit of quantification for the assay was defined as the mini mum concentration of quality controls. Results Linearity Calibration curves (n = 9) ope rating in the range of 5-500 g/mL for all four artichoke components were linear (r2 > 0.999) (Figure 2-1). Sensitivity In this study, the limit of quantification (LLOQ) is defined as the lowest concentration for quality control with an accur acy and precision better than 20 %. The LLOQ of chlorogenic acid, cynarin, luteolin-7-O-glucosi de and luteolin-7-O-glucuronide were 0.5, 0.5, 1 and 5 g/mL, respectively. Specificity The methods provided good resolutions between chlorogenic acid, cynarin, luteolin-7-Oglucoside and luteolin-7 -O-glucuronide. Peaks of all test compounds had similar retention times and the UV spectra (200 400 nm) when compared to the st andards. The wavelengths 350 and 330 nm used to quantify caffeolyquinic acids a nd luteolin derivatives at their maximum absorption, respectively, were confirmed by th eir UV spectra (Figur e 2-2). There was no endogenous interference from articho ke extract (Figure 2-3) in th is assay, indicating specificity of the methods to the tested compounds. Additio nally, The UV spectra of all tested compounds

PAGE 44

44 showed more than 99% of si milarity with those obtained using the respective standard compounds (Figure 2-3). Precision and Accuracy The precision intraand inter-day for chlor ogenic acid, cynarin, luteolin-7-O-glucoside and luteolin-7-O-glucuronide were satisfactory with CV values between 0.73 and 12.35%. Similarly the accuracy of the assay was between 94.34 a nd 107.32% for all compounds tested at three different concentrations. The results are summarized in Table 2-4. Stability The standard solutions of caffeoylquinic acid s and luteolin derivativ es were found stable on autosampler at 20oC within 24 hours (Table 2-2 and Tabl e 2-3). The shifting of the areas of each sample tested was less than 15 % of those ob tained from a fresh solution at the same level of concentrations. Quantification of Caffeoylquinic Acids (C hlorogenic Acid, Cynarin) and Luteolin Derivatives (Luteolin-7-O-glucoside, Luteo lin-7-O-glucuronide) in Artichoke Leaf Extract The results from Table 2-5 showed that th e caffeoylquinic acids were the predominant phenolic compounds of the artichok e extract, with 5-O-caffeoylquinic acid showing the highest amount. The predominant flavonoid was luteolin -7-O-glucoside, followed by luteolin-7-Oglucuronide. Discussion and Conclusion This study reported a quantitative evaluation of phenolic marker compounds of artichoke extract using a HPLC with photod iode array detector (HPLC/DAD) The identification of each compound was performed by a comparison with av ailable standards and by UV evaluation. This approach made it possible to ra pidly discriminate between caffe oyl derivatives and flavonoids. The main chemical structures of the iden tified compounds are showed in Figure 2-2 as

PAGE 45

45 chlorogenic acid, cynarin, dihydrocaffeic acid, luteolin-7-O-glucoside and luteolin-7-Oglucuronide. The HPLC profiles of the extract are shown in Figure 2-2 with a profile of the caffeoyl derivatives at 330 nm and profiles of fla vonoids at 350 nm. The quantitative HPLC/DAD findings of caffeoylquinic ester and fl avonoid are summarized in Table 2-4 The developed method is appropriate to co mpletely characterize and quantify phenolic marker compounds in artichoke extract.

PAGE 46

46 Table 2-1. Concentrations of the standard solutio ns used for the calibration curves and quality controls (QCs) of chlorogeni c acid, cynarin, lute olin-7-O-glucoside and luteolin-7-Oglucuronide Compounds Standard solutions ( g/mL)QC ( g/mL) Chlorogenic acid 5, 10, 50, 100, 40010, 25, 200 Cynarin 5, 10, 50, 100, 40010, 25, 200 Luteolin-7-O-glucoside 5, 10, 50, 100, 40010, 25, 200 Luteolin-7-O-glucucronide 5, 10, 50, 100, 250, 500 8, 75, 200

PAGE 47

47 Table 2-2. The stability test of chlorogenic ac id, cynarin and luteolin-7-O-glucoside after 24 hours on autosampler at 20oC. Data represents the percen tage remaining of all test compounds % Remaining on autosampler at 20 oC within 24 hours Compound QC1-10 g/mLQC225 g/mLQC3-200 g/mL Chlorogenic acid 90.41 2.35100.75 2.33104.58 3.19 Cynarin 95.83 10.4596.35 0.7398.58 6.10 Luteolin-7-O-glucoside 94.07 5.7298.77 5.61100.50 6.23

PAGE 48

48 Table 2-3. The stability test of luteolin-7-O -glucuronide after 24 hour s on autosampler at 20oC. Data represents the percentage remaining of all test compounds % Remaining on autosampler at 20 oC within 24 hours Compound QC1-8 g/mLQC2-75 g/mLQC3-200 g/mL Luteolin-7-O-glucuronide 90.43 7.8196.21 3.45101.53 3.52

PAGE 49

49 Table 2-4. Intra-day (n = 3) and inter-day (n = 9) assay parameters of caffeoylquinic acid (chlorogenic acid and cynarin) and luteolin derivatives (luteolin-7-O-glucoside and luteolin-7-O-glucuronide). Accuracy expre ssed as % of the theoretical concentration and precision expressed as %CV Chlorogenic acid QC1-10 g/mL QC2 g/mL QC3 g/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 5.92 12.52 11.841.8310.252.352.36 3.86 5.54 Accuracy 102.41 95.38 100.15102.24103.92100.32105.22 107.16 102.78 Inter-day QC1-10 g/mL QC2 g/mL QC3 g/mL Precision 14.67 13.167.07 Accuracy 95.21 100.56107.32 Cynarin QC1-10 g/mL QC2 g/mL QC3 g/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 6.11 9.13 10.532.1411.526.102.19 4.54 0.73 Accuracy 92.11 100.25 93.5798.23102.21102.46100.81 107.68 105.53 Inter-day QC1-10 g/mL QC2 g/mL QC3 g/mL Precision 13.31 8.738.59 Accuracy 94.34 98.90105.22 Luteolin-7-O-glucoside QC1-10 g/mL QC2 g/mL QC3 g/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 3.49 12.21 10.192.7011.776.231.42 6.67 4.52 Accuracy 102.26 97.31 106.6997.34102.65107.71106.88 104.38 109.81 Inter-day QC1-10 g/mL QC2 g/mL QC3 g/mL Precision 11.90 7.042.54 Accuracy 98.51 97.21100.53 Luteolin-7-O-glucuronide QC1-8 g/mL QC2 g/mL QC3 g/mL Intra-day Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 10.54 12.35 7.83 7.988.452.65 2.18 3.53 0.29 Accuracy 96.32 100.21 99.13 98.51102.21104.26 100.75 104.12 109.07 Inter-day QC1-8 g/mL QC2 g/mL QC3 g/mL Precision 10.19 8.725.76 Accuracy 95.54 97.19100.98

PAGE 50

50 Table 2-5. Amounts of caffeoylquinic acids and lute olin derivatives expressed as milligram per gram of dried extract Compound Amount of compounds in artichoke extract (mg/g) mean SEM 5-O-caffeoylquinic acid (chlorogenic acid) 8.71 0.59 1,3-di-O-caffeoylquini c acid (cynarin) 2.47 0.54 luteolin-7-O-glucoside 3.60 0.62 luteolin-7-O-glucuronide 2.08 0.73

PAGE 51

51 A 0 100 200 300 400 500 0 3.0106 6.0106 9.0106 1.2107 1.5107chlorogenic acid[ug/mL]AreaB 0 100 200 300 400 500 0 5.0106 1.0107 1.5107 2.0107cynarin[ug/mL]Area C 0 100 200 300 400 500 0 1.0106 2.0106 3.0106 4.0106 5.0106 6.0106 7.0106 8.0106Luteolin-7-0-glucoside[ug/mL]AreaD 0 100 200 300 400 500 600 0 1.0106 2.0106 3.0106 4.0106 5.0106 6.0106 7.0106Luteolin-7-0-glucuronide[ug/mL]Area Figure 2-1. Mean calibration curves of compounds in artichoke (n = 9). A) Chlorogenic acid. B) Cynarin. C) Luteolin-7-O-glucoside. D) Luteolin-7-O-glucuronide in methanol. Vertical bars represent the standard deviations (SD) of the means. Y = 37766 X 209223 R2 = 0.999 Y = 30030 X 45570 R2 = 0.9992 Y = 11951 X 86968 R2 = 0.9992 Y = 12680 X 7939.7 R2 = 0.9997

PAGE 52

52 Figure 2-2. HPLC separation and absorbance-wavelength spectra of chlorogenic acid, cynarin, dihydrocaffeic acid, luteo lin-7-O-glucoside and luteolin-7-O-glucuronide. Minutes 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 mVolts 0 50 100 150 200 250 300 350 400 450 Chlorogenic acid Cynarin nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 500 1000 1500 mAu 0 500 1000 1500 25.20 Min / Smooth nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 500 1000 1500 2000 mAu 0 500 1000 1500 2000 38.35 Min / Smooth nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 250 500 750 1000 mAu 0 250 500 750 1000 52.88 Min / Smooth Luteolin-7-O-glucuronide nm 200 220 240 260 280 300 320 340 360 380 400 mAu -6 -4 -2 0 2 4 6 8 10 mAu -6 -4 -2 0 2 4 6 8 10 25.63 Min / Smooth Dihydrocaffeic acid Luteolin-7-O-glucoside nm 200 220 240 260 280 300 320 340 360 380 400 mAu 0 200 400 600 mAu 0 200 400 600 51.33 Min / Smooth

PAGE 53

53 A Similarity: 0.9998 nm 200 250 300 350 400 m A u -20 0 20 40 60 80 100 120 140 160 180 200 220 240 m A u -20 0 20 40 60 80 100 120 140 160 180 200 220 240 Unknown Known B Similarity: 0.9963 nm 200 250 300 350 400 m A u 0 10 20 30 40 50 60 70 80 m A u 0 10 20 30 40 50 60 70 80 Unknown Known C Similarity: 0.9807 nm 200 250 300 350 400 m A u 0 10 20 30 40 50 60 70 80 90 m A u 0 10 20 30 40 50 60 70 80 90 Unknown Known D Similarity: 0.9753 nm 200 250 300 350 400 m A u 0 5 10 15 20 25 30 35 40 45 50 m A u 0 5 10 15 20 25 30 35 40 45 50 Unknown Known Figure 2-3. Absorbance-wavelength spectras. A) Chlorogenic acid. B) Cynarin. C) Luteolin-7-Oglucoside. D) Luteolin-7-O-glucuronide. (1 ) Represents the spectra of the standard compound and (2) represents the spectra of th e peak with same re tention time of the corresponding standard but obtained afte r injection of ar tichoke extract. 1 2 1 2 1 2 1 2

PAGE 54

54 CHAPTER 3 EFFECT OF ARTICHOKE LEAF EXTRAC T, CAFFEIC ACID DERIVATIVES AND FLAVONOIDS ON XANTHINE OX IDASE INHIBITO RY ACTIVITY Background. Xanthine oxidase (XO) is a key enzyme th at catalyses the oxidation of oxypurines (hypoxanthine and xanthine) to ur ic acid in the purine metabolic pathway [67]. The uric acid plays a vital role in producing hyperuricemia and gout. Allo purinol is a clinically used XO inhibitor in the treatment of gout. However, due to the unwanted side effect of allopurinol such as hypersensitivity problem [74], Steven-Johnson syndrome [83], renal toxicity [84], and fatal liver necrosis [85] the alternative treatment with increased therapeutic activity and less side effects is necessary. The leaves of artichoke consists of many chemical components such as caffeoylquinic acids and flavonoids and one or more of thes e components may be effective agents as XO inhibitors. Flavonoids have been shown to be inhibitors of XO activity in vitro [65]. In this aim, the efficacy of artichoke leaf ex tract and its main components in inhibiting XO was performed. Additionally, various flavonoids such as fla vones, flavonols and flavanones have been investigated as XO inhibitors. Th e results are shown in Table 3-1. Specific Aim Determine the in vitro XO inhibition of Cynara scolymus L., its compounds and some flavonoids. Materials and Methods Materials Water extract of artichoke leaf (Cynara scolymus L.) were obtained from a German extract manufacturing company (Finzelb erg, Andernach, Germany). A llopurinol, chlorogenic acid ( 95%), quercetin dihydrate (> 98%) and xanthine oxidase from bovine milk (25 units/1.3 mL),

PAGE 55

55 NaH2PO4.H2O, Na2HPO4.12H2O were purchased from Sigma Ch emical Company (St. Louis, MO, USA). Apigenin (98%), eriodictyol ( 95%), dihydrocaffeic acid (9 0-95%), luteolin (99%), luteolin-7-O-glucoside (> 90%) were purchas ed from Indofine Chemical Company, Inc. (Somerville, NJ, USA). Cynarin and naringenin ( 96%) were purchased from Carl Roth GmbH+Co. (Germany). Kaempferol (RG) was pur chased from Chromadex (Santa Ana, CA, USA). Xanthine was purchased from Carl MP Biomedicals (Solon, OH, USA). All buffers and aqueous solutions were prepared with deioni zed water obtained from a NANOPure system from Barnstead (Dubuque, IA, USA). Preparation of Working Solutions and Test Solutions Phosphate buffer solution : (A). 0.2 M NaH2PO4 solutions: NaH2PO4.H2O (2.78 g) was dissolved in distilled water to make 100.0 mL solution. (B). 0.2 M Na2HPO4 solution: Na2HPO4.12H2O (71.50 g) was dissolved in distilled wa ter to make 100.0 mL solution. 85 mL of A solution and 915 mL of B solution were added to 1000.0 mL of distilled water to make 0.1 M phosphate buffer solution, pH 7.8. Xanthine buffer solution : Xanthine (12.20 mg.) was initially dissolved in 0.25 N NaOH and then diluted with 0.1 mM phosphate buffer to obtain a 400 M solution. Xanthine oxidase : The xanthine oxidase solution wa s prepared by diluting xanthine oxidase from cows milk to a final concentra tion of 0.4 U/mL in cold 0.1 mM phosphate buffer (pH 7.8). (Enzyme 200.0 L filled up to 10.0 mL) Test solution : 1.36 mg of Allopurinol (M.W. = 136.1) was dissolved in 2000 L of DMSO to make a concentration of 50 mM so lution, which was then diluted with 0.1 mM phosphate buffer to obtain a 400, 200, 100, 50, 25, 10, 5, and 1 M solution. Apigenin, caffeic acid, chlorogenic acid, cynarin, dihy drocaffeic acid, eriodictyol, k aempferol, luteolin, luteolin-7-

PAGE 56

56 O-glucoside, luteolin-7-O-glucuronide, quercetin, naringenin were prepared in the same way as allopurinol. The final concentra tion of DMSO was less than 2%. Artichoke extract was dissolved in 1 mM phosphate bu ffer to make a concentration of 1000, 500, 300, 100 g extracts/ mL phosphate buffer. Assay Procedure for Xanthine Oxidase Inhibitions The inhibitory activity of each compound was de termined using a slight modification of the reference methods [64, 86-88]. Control: 7.0 L of xanthine oxidase buffer solution (0.4 U/mL) were added to 0.1 M phosphate buffer (127.0 L) and the mixture was incubated at 37 C for 10 minute Then 66.0 L of 400 M xanthine buffer solution were added to th e mixture and the absorbance at 295 nm of the reaction mixture was measured at 37 C for 10 min by multi-det ection microplate reader (Synergy HT). The blank solution was prepared in an analogous way, but instead of the enzyme, it contained 7 L of phosphate buffer solution. The test was performed in triplicate. Sample test: 7.0 L of xanthine oxidase buffer soluti on (0.4 U/mL) was added to a solution consisting of 0.1 M phosphate buffer pH 7.8 (77.0 L) and 50.0 L each of test samples which was treated in the same way as the control. 3.5 L of phosphate buffer solution were used instead of xanthine oxidase soluti on (0.4 U) for blank tests. Xanthine oxidase activity was expressed as percent inhibition of xanthine oxidase, calculate as (1-B/A) x 1 00, where A is the change in absorb ance of the assay without the test samples. ( abs with enzyme abs without enzyme), and B is the change in absobance of the assay with the test sample ( abs with enzyme abs without enzyme). IC50 was calculated using Graphpad Prism (version 4.0, Sandiego, CA).

PAGE 57

57 LineweaverBurk Plot Enzyme kinetics was similar to xanthine oxidase assay methodology with varying concentrations of xanthine as the substrate as 200, 150, 110, 100, 90, 80, 70, 60, and 50 M. Lineweaver-Burk plots were ge nerated in Graphpad Prism (version 4.0, Sandiego, CA). Vmax (a maximal velocity) and Km (a concentration at 50% Vmax) were calculated. Results Xanthine Oxidase Inhibitory Activity of Artichoke Extract The artichoke extract was assayed for xanthi ne oxidase inhibitory activity at 1000, 500, 300, 100 g water extracts / mL (Table 3-2). Arti choke extracts showed a dose dependence XO inhibitory effect with minimal XO inhibitory activ ity (< 5%) at 100 g / ml. Xanthine Oxidase Inhibitory Activity of Va rious Flavonoids and Compounds in Artichoke The inhibition of xanthine oxi dase results in a decreased production of uric acid was measured spectrophotometri cally at 295 nm. The IC50 values (50% inhibitory concentrations) of caffeic acid derivatives and flavonoi ds were calculated and listed in Table 3-3 and Figure 3-1 Caffeic acid and caffeic acid derivatives such as dihydrocaffeic aci d, chlorogenicacid and cynarin showed weak xanthine ox idase inhibitory effect with IC50 > 100 M. For flavonoids, only flavones (luteolin, apigenin) and flavonols (kaempferol, quercetin) were shown to have potent xanthine oxidase inhi bitory activities, with IC50 values 1.49, 2.37, 3.35, and 2.34 M, respectively. Flavanones such as naringenin a nd eriodictyol showed w eak xanthine oxidase inhibitory activity with IC50 > 50 M. Flavonoid glycosides such as luteolin-7-O-glucoside showed weaker activities with IC50 value 19.90 M.

PAGE 58

58 Inhibition Mechanism Kinetic analysis using Lineweaver-Burk plots (Figure 3-2 and Table 3-4) revealed that apigenin, luteolin and kaempferol had a linear mi xed-type mode of inhibition, as can be seen from different Vmax and different Km value. Quercetin showed a competitive type inhibition, as can be concluded from similar Vmax and different Km values. Discussion and Conclusion Artichoke leaf extract inhibited XO in vitro in a dose-dependent manner with minimal XO inhibitory activity (< 5%) at 100 g/mL as shown in Table 3-1. To our knowledge this is the first time that the XO activity of artichoke had been observed. The XO inhibitory of caffeoylquinic acids a nd flavonoids were shown in Table 3-3. and Figure 3-1. Caffeic acid and caffeic acid derivatives such as dihydrocaffeic acid, chlorogenic acid and cynarin showed weak XO inhibitory effect with IC50 > 100 M. Our results were similar to the previous study. Chan et al. [86] found hydrocaffeic acid was inactive, caffeic acid and chlorogenic acid had IC50 values about 74.6 11.04 M and 126.28 2.86 M, respectively. Nguyen et al.[89] reported IC50 value of 85.4 M by caffeic acid. The activity of flavonoids as i nhibitors of xanthine oxidase in vitro has been reported to be largely determined by the double bond between C-2 and C-3. Additionally, the absence of a hydroxyl group at C-3 enhances slightly the inhi bition effect on xanthi ne oxidase [65, 87, 90]. Our results are in agreement with these obser vations (Figure 3-1). Flavonoid aglycons, only flavones and flavonols showed potent XO inhibitory activities. The IC50 values of luteolin, apigenin, kaempferol and quercetin were 1.49, 2.37, 3.35, and 2.34 M, respectively. Flavanones such as naringenin and eri odictyol showed weak XO i nhibitory activity with IC50 > 50 M.

PAGE 59

59 Flavonoid glycosides showed a much lower in hibition of xanthine oxidase than flavonoid aglycons, such as luteolin-7-O-glucoside (IC50 = 19.90 M). This result might be due to the steric interactions of glycosides on xanthine oxidase [65, 90, 91]. The Lineweaver-Burk plot of apigenin, lute olin and kaempferol showed mixed-type inhibition. Quercetin showed a competitive type inhibition. The different types of inhibition by flavonoids have been reported in the previous studies. Lin et al. [92] reported competitive inhibition by apigenin and quercetin. Cotelle et al. [40] reported competitive inhibition by luteolin. Nguyen et al. [89] reported competitive inhibition by luteolin and apigenin. The differences observed between thes e studies could be e xplained by the different reaction mixtures, the different concentrations of enzyme and th e different methods. However, Nagao et al. [65] reported mixed-typed inhibition by luteolin and kaempferol. Van H oorn et al. [87] and Chang et al. [93] reported competitive i nhibition by quercetin. Noro et al. [94] reported mixed-typed inhibition by luteolin and apigen in. These reports are consistent with our results. The results suggest that luteolin, kaempferol and apigenin inhibit XO activity not only by competitive mode, but also by interaction with the enzyme at a site other than the active center. The significant inhibition of XO by the flavonoids in vitro suggested that they may suppress the production of active oxygen species in vivo under the conditions that XO works. Additionally, their IC50 values are comparable to that of allopurinol (3.65 M), a therapeutic drug for treating gout, which indicated a possibil ity of flavonoids for treating gout. However, recent studies have shown that after orally ad ministration of quercetin in human, most of quercetin was found in a form of metabolites in plasma [95]. Ou r study had shown that luteolin7-O-glucuronide, one of the metabolites of lu teolin, showed weaker XO inhibitory (IC50 = 20.24 M) comparing with luteolin (IC50 = 1.49 M). This result presumably indicates a weaker XO

PAGE 60

60 inhibitory activity of metabolites in vivo. However luteolin-7-O-glu curonide is not the only metabolites found in plasma after incubation of lu teolin with microsomal samples from human intestine [96]. Therefore, the in vitro data obtained from the study does not necessarily predict the in vivo effects of flavonoids on XO, and further st udy of the inhibitory effects by flavonoids in vivo will be required. In this study, artichoke extrac t, caffeoylquinic acids and various flavonoids were evaluated for the inhibition of XO activity. The extrac t and caffeoylquinic acids showed weak XO inhibition. Flavone and flavonols had a highly inhibitory effect on XO. The in vivo effect of these compounds on urate accumulation by XO remains to be studied to clarif y the roles of these compounds in human health.

PAGE 61

61 Table 3-1. Structures of various flavonoids Flavones Flavonols Flavanones 4 8 5 7 6 2 3O O OH O H4' 5' 3' 6' 2'OH O O OH O H OH OH O O OH O H OH OH Apigenin Kaempferol Eriodictyol O O OH O H OH OH OH R=H, Luteolin R=Glc, Luteolin-7-O-glucoside Quercetin Naringenin O O OH OH OH RO O O OH O H OH A B C

PAGE 62

62 Table 3-2. Results of the % XO inhibi tion screening of artichoke extract XO inhibition (%) Test Sample 100 g/mL300 g/mL500 g/mL1000 g/mL Artichoke extract 5.11 1.779.83 2.0719.76 0.8926.02 1.81 Control 1 M10 M50 M100 M Allopurinol 5.35 0.7733.50 1.1893.22 0.1997.192 0.28 Note: Data are expressed as mean SEM.

PAGE 63

63 Table 3-3. The IC50 values ( M) of test samples on xa nthine oxidase inhibition Compounds IC50 ( M) C.I. Caffeic acid and Caffeoylquinic acid Caffeic acid > 100 Cynarin > 100 Chlorogenic acid > 100 Dihydrocaffeic acid > 100 Flavonoids Flavone Apigenin 2.371.51 to 3.70 Luteolin 1.491.23 to 1.83 Luteolin-7-O-glucoside 19.9017.97 to 22.09 Luteolin-7-O-glucuronide 20.2418.47 to 22.17 Flavonol Quercetin 2.34 2.11 to 2.59 Kaempferol 3.35 2.71 to 4.14 Flavanone Naringenin 50 Eriodictyol 50 Control Allopurinol 3.653.38 to 3.72 Note: Data are expressed as mean with 95% of confidence interval (C.I.).

PAGE 64

64 Table 3-4. Vmax and Km of flavonoids on xanthi ne oxidase inhibition Compounds Vmax ( M / min) Mean SEM Km ( M) Mean SEM Type of Inhibition Control 6.27 0.358.18 2.05 Apigenin 0.5 M 3.78 0.1417.84 1.87 Mixed Luteolin 0.5 M 2.78 0.0721.10 1.37 Mixed Quercetin 0.5 M 6.22 0.3421.38 2.81 Competitive Kaempferol 0.5 M 4.50 0.3423.26 4.04 Mixed Note: Vmax is a maximum velocity; Km is a concentration at 50% Vmax.

PAGE 65

65 10-1 100 101 102 0 25 50 75 100 125 Apigenin ( M)%uric acid Formation 10-1 100 101 102 0 25 50 75 100 125 Luteolin ( M)%uric acid Formation 10-1 100 101 102 103 0 25 50 75 100 125 Luteolin-7-0-glucoside ( M)%Uric Acid Foramation 100 101 102 103 0 25 50 75 100 125 Luteolin-7-0-glucuronide ( M)%uric acid Formation 10-1 100 101 102 103 0 25 50 75 100 125 Quercetin ( M)%uric acid Formation 10-1 100 101 102 0 25 50 75 100 125 Kaempferol ( M)%uric acid Formation 10-1 100 101 102 103 0 25 50 75 100 125 Allopurinol ( M)%uric acid Formation Figure 3-1. Inhibition dose-respons e effects. A) Apigenin. B) Luteolin. C) Luteolin-7-Oglucoside. D) Quercetin. E) Kaempferol. F) Allopurinol. Data are expressed as mean SEM (n = 3). The IC50 values of each compound and their respective 95% of confidence interval (C.I.) were estimat ed by nonlinear regression using GraphPad Prism 4.0 as described in Material and Methods. G F A B C D E IC50 = 2.37 C.I. = 1.51-3.70 IC50 = 2.34 C.I. = 2.11-2.59 IC50 = 1.49 C.I. = 1.23-1.83 IC50 = 20.24 C.I. = 18.47-22.17 IC50 = 3.65 C.I. = 3.38-3.72 IC50 = 3.35 C.I. = 2.71-4.14 IC50 = 19.90 C.I. = 17.97-22.09

PAGE 66

66 -0.15 -0.10 -0.05 -0.00 0.05 0.10 0.15 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4kaempferol 0.5 M Luteolin 0.5 M control apigenin 0.5 M quercetin 0.5 M 1/[xanthine] M1/v (M/min) Figure 3-2. Lineweaver-Burk plot s in the absence (control, ) and in the presence of luteolin (0.5 M, ), apigenin (0.5 M, ), kaempferol (0.5 M, ) and quecetin (0.5 M, ) with xanthine as the substrate.

PAGE 67

67 CHAPTER 4 EFFECTS OF ARTICHOKE LEAF EXTRAC T AND VARIOUS FLAVO NOIDS ON SERUM URIC ACID LEVELS IN OXONATE-INDUCED RATS Background The previous in vitro study of XO inhibitory activity of artichoke and its components has shown that the extract and caffeic acid deriva tives had weak inhibitory activity on XO. Flavonoids such as flavone and flavonols s howed a highly inhibito ry effect with IC50 < 20 M. However, the in vivo effect of artichoke extract and its components on urates accumulation by XO is limited. Therefore, in this study, the effect of artichoke extract, and various flavonoids on serum uric acid levels in oxonate induced rats was performed. Most species, except humans, some apes and the dalmatian dogs have rather low blood levels of uric acid because of the presence of th e uric acid catabolizing enzyme uricase in the plasma and liver. Uricase transforms uric acid to allantoin, which is wa ter soluble and can be excreted [67]. Thus, in rat experiment, an uri case inhibitor such as potassium oxonate was used in order to increase endogenous ly synthesized uric acid. Specific Aim Investigate the hypouricemic activity of artic hoke leaf extract, and various flavonoids. Materials and Methods Materials Water extract of artichoke leaf (Cynara scolymus L. ) were obtained from a German extract manufacturing company (Fin zelberg, Andernach, Germa ny). Allopurinol, CMC-Na, NaH2PO4.H2O and propylene glycol were purchased fr om Sigma Chemical Company (St. Louis, MO, USA). Apigenin (98%), eriodictyol ( 95%), dihydrocaffeic acid (9 0-95%), luteolin (99%), luteolin-7-O-glucoside (> 90%) were purchas ed from Indofine Chemical Company, Inc. (Somerville, NJ, USA). Naringenin ( 96%) were purchased from Carl Roth GmbH+Co.

PAGE 68

68 (Germany). Kaempferol (RG) was purchased fr om Chromadex (Santa Ana, CA, USA). All buffers and aqueous solutions were prepared wi th deionized water obtai ned from a NANOPure system from Barnstead (Dubuque, IA, USA). Stock Solutions and Preparation of Calibration Standards Uric acid stock solution : The amount of 20.0 mg of ur ic acid (MW = 580.53 g/mol) was accurately weighed, and transferre d quantitatively to a 200.0 mL volumetric flask. The standard was then dissolved in 20.0 mL of 0.25 N Na OH, the volume was completed with phosphate buffer (pH 2.3), and the final solution mixed thor oughly. The final concentration of uric acid was 100 g/mL. Standard solutions of uric acid : From the uric acid stoc k solution, six different concentrations of standard solutions of uric acid and three quality controls (QC) were prepared in phosphate buffer pH 2.3 according Table 4-1. A ll solutions were filtered through a 0.45 m PVDF membrane filter (Milli pore Corp.) before analysis. Animals and Experimental Protocols Animals Male Sprague Dawley rats (250-350 g.) we re purchased from Harlan (IN, USA) and divided into the experimental groups; containing 8-10 rats pe r group. They were housed in plastic cages. They were allowed one week to adapt to their environment before used for experiments. All the animals were maintained on a 12hr/12hr light/dark cy cle with light on at 6 am. They were given standard chow and water ad libitum during the course of the study. All animal experiments were performed according to the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Un iversity of Florida, Gainesville, USA (NIH publication # 85-23).

PAGE 69

69 Animal model of hyperuricemia in rats Potassium oxonate (250 mg/kg) was injected intraperitoneally as a suspension in 0.8 % carboxymethyl cellulose sodium salt (CMC-Na) 1 h before orally or intraperitoneal administration of tested compounds as descri bed as follows. [97, 98] The animals were anaesthetized with halothane and blood samples (1000 L) were taken from the sublingual vein 1 h. after compound administration. Afte r the blood collection, approximately 1000 L of isotonic saline were given intr aperitoneally in order to maintain the blood fluid. The blood was allowed to clot for approximately 1 h. at room temperature and then cen trifuged at 2800 x g for 15 min. to obtain the serum which was stored at -20 C until use. Drug Administration: 1. Oral administration Artichoke leaf extract: artichoke extract (250, 500, 1000 mg/kg) and allopurinol (50 mg/kg) were dissolved in 0.8% CMC-Na. Th e control groups received 0.8% CMC-Na. The volume of the drug administered to each rats was based on the body weight of the animal measured immediately prior to each dose. The extr act and allopurinol were administered orally 1 h after the administration of pot assium oxonate. For chronic treatment, test samples were given orally once daily for 1, 3, 5, and 7 days. Flavonoids: Luteolin (16, 32, 50, 100 mg/kg: test the optimum dose), other flavonoids (50, 100 mg/kg) and allopurinol (50 mg/kg) were administered oral ly at one hour after potassium oxonate. Flavonoids and allopurinol were suspended in 1:1 (propyl ene glycol (PG): water). The control groups received 1:1 (PG: water). The vo lume of the drug administered to each rats was based on the body weight of the animal measured immediately prior to each dose.

PAGE 70

70 2. Intraperitoneal administration Artichoke (500 mg/kg), caffeoylqui nic acids (50mg/kg) such as caffeic acid, chlorogenic acid, cynarin, and flavonoids (50mg/kg) such as luteolin, apigenin, querc etin and allopurinol (50mg/kg) were administered in traperitoneal injection (i.p.) at one hour after potassium oxonate. Artichoke extract were dissolved in 0.8% CMC-Na. Other test samples were suspended in 1:1 (PG: water). The control groups received 1:1 (PG: water). Uric Acid Assay Uric acid in rat serum was determined by reversed-phase high performance liquid chromatography and photodiode array detection ( DAD) [99] [100].Standards were prepared by diluting the stock uric acid solution with 200 mM phosphate buffer (0.1 mg/mL). The stock solution was prepared as follows. Uric acid has lo w solubility in water. Therefore, an aliquot (20.0 mL) of a 0.25 N NaOH was dropped into 20.0 mg of uric acid. Sonicate shortly and fill up with buffer to 200.0 mL. Samples were analyzed us ing a reverse-phase part ition mode of HPLC with diode array detector A Shimadzu VP series HPLC system (Kyoto, Japan) equiped with an SPD-M10Avp diode array detector was used for this work. A Lichrospher 100 RP-18 (5 m. Merck KgaA) was used for the separation of ur ic acid. The column temperature was kept at 25oC. The mobile phase was 200 mM phosphate buffer (NaH2PO4, pH 2.0). The flow rate was 0.5 mL/min. Ten micro liters of each sample was injected into the RP-HPLC system. Comparing the respective peak area in the chromatogram w ith the value from a standard calibration curve quantitated uric acid. Preparation of Rat Serum The proteins in rat plasma were precipitate by adding 150.0 L of 10% trichloroacetic acid to 150.0 L of plasma and adding 750.0 L of buffer to make 1 mL The precipitates were

PAGE 71

71 removed from the mixture by centrifugation at 3, 000 g for 3 min. Supernatants were filtered through 0.45 m filters and ten micro liters of the plas ma sample were injected into the RPHPLC with photodiode array detector system. Statistical Analysis All data are expressed as the mean SEM. Group mean differences were ascertained with analysis of variance (ANOVA). Multiple compar isons among treatment means were checked with the Tukeys test. The results were considered significant if the probability of error was < 0.05 Validation The method was validated over the range of con centration of uric acid present in serum. The validation parameters of linearity, sensitivit y, specificity, precision, accuracy and stability were determined. The linearity of the calibration curves was de termined by least-squares linear regression method and expressed in terms of coefficient of determination (r2). The intraand inter-day precision and accuracy were measured by triplicate analyses of three different concentration levels (low, medium and high) of quality cont rol standards on the same day and on different days. The precision was based on the calculation coefficient of variation (CV %), and the accuracy was defined as the percent difference be tween the theoretical and measured values. The limit of quantification for the assay was defined as the mini mum concentration of quality controls. 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%.

PAGE 72

72 Results Validation of Analytical Method to Measure Uric Acid in Rat Serum. Linearity Calibration curves (n = 9) ope rating in the range of 2-50 g/mL for uric acid were linear (r2> 0.999) (Figure 4-1). Sensitivity In this study, the limit of quantification (LLOQ) is defined as the lowest concentration for quality control. This concentration would be acceptable with the precision (%CV < 20), and accuracy (%error < 20).The LLOQ of uric acid in plasma was 0.1 g/mL. Specificity The method provided good resolutio ns between uric acid and in terference in serum. Peak of uric acid had similar retention times to the standard. The method showed good specificity since the chromatograms of the serum samples di d not show any co-eluting peak with similar retention time as uric acid as shown in Figure 4-2. Precision, accuracy and recovery The precisions intraand inter-day for uric aci d were satisfactory with CV values between 1.2 and 12.3%. Similarly, the accuracy of the assay obtained with quality control samples containing 3, 12, and 35 g/mL uric acid was between 91.9 and 109.1% of the nominal values. The mean recovery assessed at three distin ct levels of concentration (1, 10 and 20 g/mL) ranged from 92.8 to 94.7% of the expected values. The results are summarized in Table 4-2. Stability Uric acid was stable under th e tested conditions. The mean % remaining in rat serum after 2 hours at room temperature was 98.1 6.2, 100.4 5.7, 100.3 2.8 for the low, medium and

PAGE 73

73 high concentrations, respectively. Uric acid was stable on autosampler at 20 oC within 24 hours (Table 4-3). Effect of Artichoke Extract and Its Compound s on Serum Urate Levels in Hyperuricemic Rats Oral administration of ar tichoke in acute treatment Uricase inhibitor potassium oxona te treatment showed hyperuricemia in rats, as indicated by increased in serum uric acid levels from 9.76 to 33.40 g/mL (Table 4-4 and Figure 4-3). Artichoke water extracts did not affect the serum uric acid level after 1 h treatment. In contrast, allopurinol (50 mg/kg) lowe red the uric acid levels in hyperuricemic rats. Oral administration of arti choke in chronic treatment The hypouricemic effects of the orally administ ered artichoke water extracts on serum uric acid levels in oxonate-petreated rats are shown in Table 4-5 a nd Figure 4-4. After day 1, 3, 5 and 7 treatment, when compared with that of the hyperuricemic control group, artichoke water extract did not show effect on serum uric acid le vels. In contrast, allopurinol (50 mg/kg) lowered the uric acid levels in hyperuricemic rats after da y 1, 3, 5 and 7 treatments. Oral administration of compounds in artich oke and various flavonoids in acute treatment Potassium oxonate treatment caused hyperuricemi a in rats, as indicated by increase in serum uric acid levels. As shown in Table 4-6, Figure 4-5, Figure 4-6 and Figure 4-7, luteolin at dose 16 and 32 mg/kg did not show a decrease in serum urate levels compared with the control group. Therefore, the higher doses such as 50 mg/kg and 100 mg/kg were performed for all flavonoids. The results showed that at 50 and 100 mg/kg, apigenin, eriodict yol, luteolin, luteolin7-O-glucoside, naringenin, and quercetin did not affect the serum uric ac id level as shown in Table 4-6, Figure 4-6, and Figure 410. In contrast, the referen ce drug allopurinol (50 mg/kg) significantly lowered the uric acid levels in hyperuricemic rats.

PAGE 74

74 Intraperitoneal administrati on of artichoke, compounds in artichoke and various flavonoids in acute treatment The hypouricemic effects of the intraperitoneal treatment of artichoke water extracts, apigenin, eriodictyol, luteolin, luteolin-7-O-g lucoside, naringenin and quercetin on serum uric acid levels in oxonate-pretreated ra ts are shown in Table 4-7 and Figure 4-8. After 1 h treatment, when compared with that of the hyperuricemic control group, none of the test samples showed effect on serum uric acid levels In contrast, allopurinol ( 50 mg/kg) was active in this experiment. Discussion and Conclusion Our previous data showed that flavonoids c ould inhibit the formation of uric acid from xanthine by XO in vitro Thus, there is a possibility that the flavonoids may inhibit the XO in vivo which results in the decrease of uric acid levels in plasma. Uricase inhibitor such as potassium oxonate is needed to perform the experi ment in rats. Potassium oxonate is an inhibitor of uricase. An i.p. injection of oxonate could pa rtially block the convers ion of uric acid to allantion and thus ar tificially elevate the plasma uric acid level in rats to provide a hyperuricemic animal model [101, 102]. Howeve r, in the present study, the in vivo experiments demonstrated that artichoke leaf extract (250,500 and 1000 mg/kg) and flavonoi ds (50 and 100 mg/kg) could not exert hypouricemic in the oxonate-induced rats after 1 h oral administration. This lack of effect via the oral route might be due to the first pass metabolism through gut, intestine or liver as reported in previous studies. In vitro experiments demonstrated that 74% of luteolin was conjugated to glucuronic acid af ter incubation with microsomal samples from human intestine [96]. Wittemer et al [30] had shown that after oral admi nistration of artichoke leaf extract in human, none of the genuine extract constituents such as caffeoylquinic acids (e.g. chlorogenic acid, cynarin), caffeic acid and flavonoids (e.g. lu teolin-7-O-glucosides) could be detected in

PAGE 75

75 plasma and urine, however, the metabolites in the form of glucuronides and sulfates were observed instead. Chen et al. [95] reported the sy stemic bioavailability of quercetin and quercetin conjugates as 5.3% and 55.8%, respectively in rats after administration of quercetin. Additionally, about 93.3% of quercetin was metabolized in th e gut, with only 3.1% metabolized in the liver. Beside the first pass effect, the absorption of the compound should also be considered. In humans, peak plasma concentrations of total luteolin and total caffeic acid were reached within 0.5 h and 1 h respectively, after a single oral do se of artichoke leaf extract (153.8 mg) [30]. After the flavonol administration in human, the p eak in blood occurred at approximately 2.9 h [103]. In rats, flavonoids seemed to appear more rapidly. Luteolin appeared in plasma 15 min after given via gastric intubation [104]. Apigenin occurred in plasma 30 mi n after intraperitoneal administration in rats [105]. These results from the literature suggest ed a relatively rapid absorption of flavonoids. Thus, in our study, mo st of flavonoids might be metabolized and excreted after 1 h oral ad ministration of flavonoids. It has been reported that in traperitoneal administration of some flavonoids such as apigenin, kaempferol, naringenin and rutin significan tly reduced small and large intestinal transit in mice [106]. The compounds via oral route have both intestinal absorpti on and first pass effect through the liver. Thus, the amount of active com pounds that appear via oral route may be higher than those via intraperitoneal route because of different absorption. In our study, after i.p. injection of artichoke leaf extr acts (500 mg/kg), flavonoids (50 mg/kg) and caffeoyl quinic acids (50 mg/kg) to the hyperuricemic rats, they did no t elicit any significant hy pouricemic effect. This lack of effect via intraperitoneal route might be due to small and large intestinal transit reduction and first pass metabolism through liver.

PAGE 76

76 Jimnex-Escrig et al. [8] found a 55 % reduction of plasma urate levels in rats fed with a diet containing the edible part of artichoke ( 138 g/kg diet) for 3 weeks. The differences observed between our study and this study could be explained by the differe nt part of artichoke (heads of artichoke), the different species of rats (Wistar rats) and the different formulation of artichoke (a diet contai ning artichoke). Zhu et al. [66] re ported a hypouricemic effect in oxonatepretreated mice after a three-time pretreatment of quercetin and rutin (100 mg/kg). The test compounds were dissolved in propyle neglycol/water (50/50). Yu et al. [107] showed a decrease of plasma urate levels after 5 h administration of morin (80 mg/kg) in hyperuricemic rats. Morin was prepared in 0.3% Tween 20. The differences observed between these studies are the species of animals and the solvent used to prepare test samples. In conclusion, the data reported in this st udy indicated that oral and intraperitoneal administration of artichoke leaf extract, fl avonoids and caffeolyquinic acids could not reduce serum urate levels of hyperuricemic rats induced by oxonate. This lacks of effect might be due to first pass metabolism through gut, intestine or liver for the oral route and due to small and large intestinal transit reduction and first pass meta bolism through liver for intraperitoneal route. Additionally, blood collection af ter 1 h treatment may not be a right time point since caffeoylquinic acids and flavonoids have been reported to have rapid absorption and metabolite. Therefore, the pharmacokinetics of flavonoids and caffeoylquinic acids should be further studied.

PAGE 77

77 Table 4-1. Concentrations of the standard solutio ns used for the calibration curves and quality controls (QCs) of uric acid Standard Uric acid stock solution (mL) Fill volume up in volumetric flask (mL) Concentration ( g/mL) 1 0.20102 2 0.2555 3 11010 4 0.75515 5 1520 6 51050 QC1 0.3103 QC2 1.21012 QC3 3.51035

PAGE 78

78 Table 4-2. Intra-day (n = 3), interday (n = 9), and recovery (n = 3) assay parameters of uric acid in rat serum. Precision expressed as CV %, accuracy and recovery as % of the theoretical concentration Intra-day QC1 3 g/mL QC2 12 g/mL QC3 35 g/mL Day 1 Day 2 Day 3Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 11.4 12.3 10.15.71.28.73.5 5.4 4.6 Accuracy 94.7 91.9 94.5100.895.398.7109.1 100.6 102.3 Inter-day QC1 3 g/mL QC2 12 g/mL QC3 35 g/mL Precision 6.5 3.43.1 Accuracy 94.3 100.4108.8 Recovery Uric acid 1 g/mL Uric acid 10 g/mL Uric acid 20 g/mL % 92.8 94.194.7 CV% 10.2 5.45.5

PAGE 79

79 Table 4-3. The stability test af ter 24 hours on autosampler at 20oC. Data represents the percentage remaining of uric acid % Remaining on autosampler Luteolin concentration 12 hours24 hours QC1-3 g/mL 95.3 6.598.6 10.2 QC2-12 g/mL 101.5 3.6100.5 5.2 QC3-35 g/mL 106.0 2.4106.7 3.1

PAGE 80

80 Table 4-4. Hypouricemic effects of allopurinol, water extract of ar tichoke on plasma urate levels ( g/mL) in oxonate-pretreated rats in acute treatment Treatment groups Animals Dosage of drugs (mg/kg) Serum Urate levels ( ug/ml) SEM Normal rats 109.76 0.95 Hyperuricemia rats dosed with vehicle (0.8% CMC-Na) 1033.40 1.64 Hyperuricemia rats dosed with artichoke extract 10250 30.56 0.77 10500 32.02 1.25 101000 32.34 1.90 Hyperuricemia rats dosed with allopurinol 1050 6.26 0.35 Note: Hyperuricemia was induced by injecting po tassium oxonate. They were then orally given artichoke extract, or allopur inol at different doses. Da ta represent mean value ( SEM) of plasma urate level ( g/mL) in animals groups (n = 10). For statistical signif icant,* indicates P < 0.001 when the compounds-treated animals we re compared with the hyperuricemic rats without drug treatment (vehicle controls).

PAGE 81

81 Table 4-5. Hypouricemic effects of allopurinol and artichoke extr act on plasma urate levels ( g/mL) in oxonate-pretreated ra ts after chronic treatment Duration of drug treatment (days) Treatment groups N Dosage (mg/kg) 1 3 5 7 Normal rats 8 15.06 0.77 18.95 0.83 11.52 0.83 14.82 1.49 Hyperuricemia rats dosed with vehicle 8 29.94 0.87 28.73 1.38 26.22 1.39 28.75 0.73 Hyperuricemia rats dosed with artichoke extract 8 500 33.38 1.19 27.87 0.65 25.94 1.34 29.67 1.37 8 1000 31.35 1.05 26.00 1.53 24.57 2.08 26.93 1.22 Hyperuricemia rats dosed with allopurinol 8 50 9.02 0.37* 9.31 0.70* 5.89 0.34* 6.10 0.8* Note: Hyperuricemia was induced by injecting po tassium oxonate. They were then orally given artichoke or allopurinol at different doses. Data represent mean value ( SEM) of plasma urate level ( g/mL) in animals groups (n = 8). For st atistical significant, indicates P < 0.001 when the compounds-treated animals were compared with the hyperuricemic rats without drug treatment (vehicle controls).

PAGE 82

82 Table 4-6. Hypouricemic effects of allopurinol, apigenin, eriodict yol, luteolin, luteolin-7-Oglucoside, naringenin, quercetin on plasma urate levels ( g/mL) in oxonate-pretreated rats after oral administration Treatment groups Animals Dosage of drugs (mg/kg) Serum Urate levels (ug/mL) mean SEM Normal rats 814.58 0.97 Hyperuricemia rats dosed with vehicle (PG:water,50:50) 828.87 1.22 Luteolin 816 26.21 1.28 832 33.14 3.42 850 27.93 1.55 8100 27.09 0.81 Luteolin-7-O-glucoside 850 30.54 1.34 8100 29.75 2.12 Apigenin 850 32.90 2.56 8100 33.99 1.72 Eriodictyol 850 27.50 0.64 8100 33.51 2.05 Kaempferol 850 29.77 0.91 8100 27.54 2.17 Naringenin 850 27.82 1.14 8100 30.45 1.80 Quercetin 850 27.28 0.84 8100 27.84 2.74 Allopurinol 850 8.53 2.091* Note: Data represent mean value ( SEM) of plasma urate level ( g/mL) in animals groups (n = 8). For statistical si gnificant, indicates P < 0.001 wh en the compounds-treated animals were compared with the hyperu ricemic rats without drug trea tment (vehicle controls)

PAGE 83

83 Table 4-7. Hypouricemic effects of allopurinol, apigenin, eriodict yol, luteolin, luteolin-7-Oglucoside, naringenin, quercetin on plasma urate levels ( g/mL) in oxonate-pretreated rats after i.p injection Treatment groups Animals Dosage of drugs (mg/kg) Serum Urate levels (ug/mlL) mean SEM Normal rats 511.06 0.84 Hyperuricemia rats 531.14 1.65 Artichoke 5500 27.32 2.11 Caffeic acid 550 35.21 3.02 Chlorogenic acid 550 33.86 3.15 Cynarin 550 35.11 4.05 Apigenin 550 34.06 2.89 Quercetin 550 26.39 0.92 Luteolin 550 27.60 2.17 Allopurinol 550 9.12 1.39* Note: The hyperuricemic rats were produced by potassium oxonate pretreatment. They were then intraperitoneally given of ar tichoke leaf extracts (500 mg/ kg), 50mg/kg of allopurinol, apigenin, eriodictyol, luteoli n, luteolin-7-O-glucoside, nari ngenin and quercetin. Data represent (mean SEM) of plasma urate level ( g/ml) in animals groups (n = 5). For statistical significant, i ndicates P < 0.001 when the com pounds-treated animals were compared with the hyperuricemic rats wit hout drug treatment (v ehicle controls)

PAGE 84

84 0 10 20 30 40 50 60 0 5.0105 1.0106 1.5106 2.0106 2.5106 3.0106 3.5106 4.0106 4.5106Uric acid [ g/mL]Area Figure 4-1. Mean calibration curves (n = 9) of uric in serum. Vertic al bars represent the standard deviations (SD) of the means. Y = 82480 X + 6314 r2 = 0.9990

PAGE 85

85 Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 mVolts-1 0 1 2 3 4 5 6 7 8 9 10 Figure 4-2. HPLC chromatogram of uric acid in serum. Uric acid

PAGE 86

86 Normal Control 250 500 1000 Allopurinol 0 10 20 30 40*Uric acid in plasma (ug/ml) Figure 4-3. Acute effects of allopur inol, artichoke extract on serum urate levels in rats pretreated with the uricase inhibitor potassium oxona te. Rats were treated with potassium oxonate (250 mg/kg) before administra tion of artichoke a nd allopurinol (50 mg/kg).The data represent the mean SEM for 10 animals. P < 0.001; significant from the control.

PAGE 87

87 Day1 0 5 10 15 20 25 30 35*Uric acid in serum(ug/ml) Day 3 0 5 10 15 20 25 30 35*Normal Control Allopurinol 50 mg/kg Artichoke500mg/kg Artichoke 1000 mg/kg Uric aicd in plasma (ug/ml) Day 5 0 5 10 15 20 25 30 35*Uric acid in serum(ug/ml) Day 7 0 5 10 15 20 25 30 35*Normal Control Allopurinol 50 mg/kg Artichoke500mg/kg Artichoke 1000 mg/kg Uric acid in serum (ug/ml) Figure 4-4. Chronic effects of a llopurinol, artichoke extratc on serum urate levels in oxonatetreated rats. Rats were treated with potassium oxonate (250 mg/kg) before administration of artichoke (500, 1000 mg/kg) and allopuri nol (50 mg/kg) for 1, 3, 5 and 7 days. The data represent the mean SEM for 8 animals. P < 0.001: significantly from the control.

PAGE 88

88 NormalControlAllopurinol163250100 0 10 20 30 40 Luteolin (mg/kg)*Uric acid levels in serum (g/ml) Figure 4-5. Effects of allopurinol an d luteolin on serum urate levels in rats pretreated with the uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before oral administration of luteolin (16, 32, 50, 100 mg/kg) and allopurinol.The data represent the mean SEM for 8 animals. *P < 0.001: significantly from the control.

PAGE 89

89 NormalControlAllopurinolApigeninL-glcKaempferolQuercetinEriodictyolNaringenin 0 10 20 30 40*Uric acid levels in serum (g/ml) Figure 4-6. Effects of allopurinol, apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, and quercetin on serum urate levels in rats pret reated with the uricase inhibitor potassium oxonate. Rats were treated with potassi um oxonate (250 mg/kg) before oral administration of 50 mg/kg of apigeni n, eriodictyol, luteo lin-7-O-glucoside, naringenin, quercetin and allopurino l. The data represent the mean SEM for 8 animals. P < 0.001: significantly from the control.

PAGE 90

90 NormalControlAllopurinolApigeninL-glcKaempferolQuercetin Eriodictyol Naringenin 0 10 20 30 40*Uric acid levels in serum (g/ml) Figure 4-7. Effects of allopurinol apigenin, eriodictyol, luteolin-7-O-glucoside, naringenin, quercetin on serum urate levels in rats pret reated with the uricase inhibitor potassium oxonate. Rats were treated with potassi um oxonate (250 mg/kg) before oral administration of 100 mg/kg of apigeni n, eriodictyol, luteol in-7-O-glucoside, naringenin, quercetin and a llopurinol (50 mg/kg).The data represent the mean SEM for 8 animals. P < 0.001: significantly from the control.

PAGE 91

91 Normal Control Artichoke Allopurinol Caffeic acidChlorogenic CynarinApigeninQuercetin Luteolin 0 10 20 30 40*Uric acid levels in serum (g/ml) Figure 4-8. Effects of artichoke extract, allopurinol, caffeic aci d, chlorogenic acid, cynarin, luteolin, apigenin and quercetin on serum ur ate levels in rats pretreated with the uricase inhibitor potassium oxonate. Rats were treated with potassium oxonate (250 mg/kg) before i.p. injection of artichoke (500 mg/kg), and 50 mg/kg of allopurinol, caffeic acid, chlorogenic acid, cynarin, lute olin, apigenin and quercetin. The data represent the mean SEM for 8 animals. P < 0.001: si gnificantly from the control.

PAGE 92

92 CHAPTER 5 THE EFFECT OF ARTICHOKE LEAF EXTRACT AND ITS COMPOUNDS ON ANTIOXIDANT ACTIVITY IN VITRO AND IN RATS Background Reactive oxygen species (ROS) su ch as superoxide anion, hydrogen peroxide and singlet oxygen are implicated in some diseases such as inflammation, cancer, ageing, and degenative diseases [108]. ROS are common products of severa l oxidative systems, es pecially the xanthinexanthine oxidase system. Xanthine oxidase is an important enzyme whic h catalyses the oxidation of hypoxanthine to xanthine and then to uric aci d in man. The accumulation of uric acid can not only lead to gout and hyperuricemia, but can al so provoke inflammation by various mechanisms such as an neutrophil recruitment and the re lease of leukotriene B4, interleukin-1 (IL-1), interleukin-2 (IL-2) and superoxi de [109, 110]. Therefore, the co mpound that can scavenge free radicals could have a beneficial effect not only in the treating of gout and hypreuricemia, but also in the alleviation of inflammation. Artichoke leaves (Cynara scolymus L .) is a good source of natural antioxidants since major compounds in artichoke leaves are polyphenolic compounds with monoand dicaffeoylquinic acids and flavonoids. Artichoke leaf extract ha s been reported to show antioxidative and protective properties against hydrope roxide-induced oxidative stress in cultured rat hepatocytes [6], to protect low density lipoprotein from oxidation in vitro [9], to inhibit hemolysis induced by hydrogen peroxide and to inhibit oxidative stress when human leuc ocytes are stimulated with agents that generate reactive oxygen species such as hydrogen peroxide [16]. The phenolic compounds in artichoke have been repor ted to show antioxidant activity in vitro [24]. However, only one study reported an effect of the edible pa rt of artichoke on biomarkers of antioxidants in rats [8]. Therefore, in vivo studies of artichoke leaves on antioxidant activity should be performed.

PAGE 93

93 In this study, the in vitro antioxidant properties of majo r compounds in artichoke extract (caffeoylquinic acids and flavonoids) and some re ference flavonoids (quercetin) as shown in Figure 5-1 were investigated. More over, the effect of the intake of artichoke extract, and its components for 2 h and 3 weeks on total antioxidant activity and antioxidant enzyme glutathione peroxidase in plasma of male rats were evaluated. Specific Aims Investigate whether artichoke extract a nd its compounds show antioxidant activity in vitro and in rats. Materials and Methods Materials Artichoke leaf extract was obtained from a German extract manufacturing company (Finzelberg, Andernach, Germany). Dihydrocaffeic ac id (90-95%), luteolin (99%), and luteolin7-O-glucoside (> 90%) were purchased from Indo fine Chemical Company, Inc. (Somerville, NJ, USA). Chlorogenic acid ( 95%), dihydrate (> 98%), dipotassium hydrogenphosphate (K2HPO4), sodium dihydrogenphosphate (NaH2PO4), and AAPH (2, 2-Azobis (2amidinopropane) dihydrochloride were purchased from Sigma Chemical Company (St. Louis, MO, USA). Cynarin was purchased from Carl Roth GmbH+Co. (Germany). Fluorescein was purchased from Fluka (Milwaukee, WI, USA). Acetonitrile (CH3CN), methanol, perchlorogenic acid (PCA), and trifluoroacetic acid (TFA) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Trolox 97% (6-Hydroxy-2, 5, 7, 8-tetram ethylchroman-2-carboxylic acid) (Biomol, PA, USA), were used. All aqueous solutions were prepared with purified water obtained from a NANOPure system from Barnstead (Dubuque, IA, USA). Fluorescence filters with an excitation wavelength of 485 nm and emission wa velength of 538nm were used. The microplate

PAGE 94

94 reader (Synergy HT) was purchased from BioTEK (Wincoski, VT, USA). The 96 well-plates (Corning) were puchased from Fisher Scientific (Fair Lawn, NJ, USA). Animals Male Sprague Dawley rats (250-350 g) we re purchased from Harlan (IN, USA) and divided into the experimental groups; containing 8 rats per group. They were housed in plastic cages. They were allowed one week to adapt to their environment before used for experiments. All the animals were maintained on a 12h/12h light/dark cycle. They were given standard chow and water ad libitum during the course of the study. All animal experiments were performed according to the policies and guidelines of the Institutional Animal Care and Use Committee (IACUC) of the University of Florida, Gainesville, USA (NIH publication # 85-23). Acute treatment Artichoke extract (500, 1000 mg/kg), luteolin (25, 50 mg/kg), and quercetin (25 mg/kg) was administered orally. Artichoke was suspende d in 0.8% carboxyl methylcellulose sodium salt (CMC-Na). The control groups received 0.8% CMCNa. The volume of the drug administered to each rats was based on the body weight of the anim al measured immediately prior to each dose. Rats were anaesthetized with halothane and 500 L blood samples were taken from sublingual vein at 2 h into tubes contai ning sodium heparin 900 units. Each tube was centrifuged at 2800 x g for 15 min to obtain the plas ma which was stored at -80 C until use for antioxidant analysis. Chronic treatment Artichoke extract (500, 1000 mg/kg), luteolin (25, 50 mg/kg), and quercetin (25 mg/kg) were administered orally once a day for 21 da ys. Artichoke and flavonoids were suspended in 0.8% carboxyl methylcellulose sodium salt (CMC -Na). The control grou ps received 0.8% CMCNa. 1000 L blood samples were taken from subli ngual vein on day 0, 7, 14 and 21. Prior to

PAGE 95

95 blood collection, rats were anaest hetized with halothane and bl ood loss was replaced with an equal volume of normal saline. Blood samples were treated in the same way as acute treatment. Plasma samples were used for antioxidant activ ity, uric acid concentr ation and glutathione peroxidase activity. Assessment of Antioxidative Capacity in Vitro and Plasma Antioxidant Status ORAC assays were carried out on a syner gy HT plate reader (Biotex, USA) with fluorescence filters (excitation wavelength: 485 nm, and emission filter: 538 nm).The temperature of the incubator was set to 37 oC. Procedures were base d on the previous report by Ou et al.[111]. Briefly; AAPH was used as peroxyl generator and Trolox as a control standard. 50.0 L of sample, blank, and Trolox calibration solutions were transformed to 98-well microplates in triplicate. 100.0 L of fluorescene solution were added and then 50 L of AAPH solution were added immediately before reading in microplate reader. Fluorescence reading were taken every 10 min for a duration of 70 min. Final results were ca lculated based on the difference in the area under the fluorescein d ecay curve between the blank and each sample. Artichoke extracts 10.0 mg were dissolved in 10.0 mL of phosphate buffer pH 7.0 and then dilute in a ratio of 1 to 100 with phosphate buffer; phenolic co mpounds were dissolved in DMSO and then diluted with phosphate buffer pH 7.0. The concentration of DMSO was less than 0.1 % for in vitro study. ORAC values were expressed as re lative Trolox equivalent s in respect to 25 M of phenolic compounds and expressed as mol Trolox quivalent ( TE)/ g of artichoke extracts. Assessment of Uric Acid in Plasma Uric acid was measured by reversed-phase high performance liquid chromatography and photodiode array detection (DAD). Standards were prepared by diluting the stock uric acid

PAGE 96

96 solution with 200 mM phosphate buffer (0.1 mg/m L). The stock solution was prepared as follows. Uric acid has low solubility in wate r. Therefore, a small amount of 0.25 N NaOH was added into uric acid. Sonicated shortly and filled up with buffer. The column temperature was kept at 25 oC. The mobile phase was 200 mM phosphate buffer (NaH2PO4, pH 2.0). The flow rate was 0.5 mL/min. Ten micro liters of each sa mple was injected into the RP-HPLC system. Comparing the respective peak area in the chromatogram with the value from a standard calibration curve quant itated uric acid. The proteins in rat plasma were precipitated by adding 150.0 L of 10% tricholoacetic acid to 150.0 L of plasma and adding 700.0 L of buffer to make 1 mL The precipitates were removed from the mixture by centrifugation at 3, 000 g for 3 min. Supernatants were filtered through 0.45 m filters and ten micro liters of the plas ma sample were injected into the RPHPLC with photodiode array detector system a nd measured at a wavelength value 285 nm. The plasma nonprotein fraction was prepared by diluting plasma with 0.5 M perchloric acid (PCA) (1:1, v/v). The samples were vorte xed for 15 sec and centrifuged at 4000 rpm for 10 min at 4 C. Then, the supernatant was removed as the plasma nonprotein fraction, and diluted in a ratio of 1 to 20 with phosphate buffer pH 7.0 for the analysis. All plasma samples were assessed within 1 week after blood drawing. OR AC values were expressed as mmol Trolox equivalents per liter. Assessment of Glutathione Peroxidase (GPx) in Plasma GPx activity was determined by using a glut athione peroxidase assay kit. (Cayman chemical company, Ann Arbor, MI, USA)

PAGE 97

97 Statistical Analysis All data are expressed as the mean SEM Group mean differences were ascertained with analysis of variance (ANOVA). Multiple compar isons among treatment means were checked with the Tukeys test. The results were considered significant if the probability of error was < 0.05 Results Antioxidant Activity in Vitro The antioxidant activities of artichoke extr act and phenolic compounds were estimated by ORAC assay as shown in Table 5-1. On e gram of artichoke extract had 1623.35 mol of Trolox equivalent. 1, 3-di-O-ca ffeoylquinic acid (cynarin), quercetin a nd luteolin showed the strongest antioxidant activity with 6.73, 5.30 a nd 5.16 relative Trolox equivalent in vitro respectively (Table 5-2). Plasma Antioxidant Activity in Vivo Acute treatment There was no significant difference in the pl asma antioxidant activity between artichoke group and the control group afte r orally treatment of artichoke (500, 1000 mg/kg), luteolin (25, 50 mg/kg) and quercetin (25 mg/kg) for 2 h as shown in Table 5-3. Chronic treatment After orally administration of 500 and 1000 mg/kg of artichoke extract for 21 days, there was no significant differen ce in the plasma antioxidant activity between the artichoke groups and the control groups. 25 and 50 mg/kg of lu teolin and 25 mg of qu ercetin also did not showed antioxidant activity in vivo as shown in Table 5-4.

PAGE 98

98 Plasma Urate Concentrations and Plasma Gl utathione Peroxidase Activity after The Treatment with Artichoke Extract and Phenolic Compounds There were no significant diffe rence in the plasma urate concentrations or glutathione peroxidase activity between the experiment groups and the control group after orally administration of 500 and 1000 mg/kg of artichoke extract, 25 and 50 mg/kg of luteolin and 25 mg of quercetin over a 21 days period as shown in Table5-5 and Table 5-6. Discussion and Conclusion The in vitro antioxidant activites were tested by using the ORAC assay, which evaluates the radical scavenging activity of the test samp les towards peroxyl radicals generated through the thermal decomposition of a radical initiator ( AAPH). Table 5-1 and Table 5-2 summarized the results expressed as mol of Trolox equivalents/ g of arichoke extract and relative Trolox equivalents for caffeic acid deriva tives and flavonoids. It showed that the extract and all the compounds were found to be more active than Tr olox.This result is consistant with previous studies. Ou et al. [111] found that caffeic aci d, chlorogenic acid and quercetin showed high relative ORAC values. Wang et al. [24] measured the re lative antioxidant act ivities (% inhibition of DPPH free radicals) of phenolic compounds and found that cynarin, cynaroside, luteolin-7rutinoside and chlorogenic acid sh owed high antioxidant activities. The antioxidant activities of phenolic com pounds have been reported to be largely determined by the number of hydroxyl groups on the aromatic ring and the position of the substituents. The higher the number of hydroxyl gr oups, the greater the antioxidant activity. In addition, the presence of a catechol group in phenolic ring also increases th e antioxidant activity [24]. Our results are in agreement with this re port (Figure 5-1). Cyna rin, with two adjacent hydroxyl groups on both phenolic rings showed th e highest antioxidant activity. Quercetin, luteolin and luteolin-7-O-glucoside with tw o adjacent hydroxyl groups on one ring and only a

PAGE 99

99 single hydroxyl group on the othe r ring showed less antioxidant activity, which was still higher than chlorogenic acid, caffeic acid and dihydro caffeic acid with two adjacent hydroxyl groups on one ring. Artichoke leaf extract co ntains caffeoylqunic acids and fl avonoids, thus, the antioxidant activity of the extr act was high (1623.36 2.84 mol TE/ g of dry extracts). In vivo antioxidant activity showed that the acute and chroni c treatment of artichoke extract (500, 1000 mg/kg), luteolin (25, 50 mg/kg) and quercetin ( 25 mg/kg) could not increase the total antioxidant activity in rats plasma. In addition, the chroni c treatment did not lead to an increase in the value of glutathi one peroxidase (a marker of an tioxidative defense), and in the uric acid levels (endogenous antio xidant compound) in plasma. This lack of effect might be due to the low absorption of caffeoylquinic acid s and flavonoids. In human study, the maximal plasma concentrations of flavonoids, reached be tween 1 and 3 h after consumption of flavonoid rich food, is between 0.06 and 7.6 M for flavonols, flavanols and flavanones [112]. In rats, the concentration of luteolin at 30 min was 15.5 3.8 nmol/mL after admi nistration of one single dose of luteolin [104]. The maximum concentration of total caffeic acids a nd luteolin, reached at 0.83 and 0.36 h, respectively, were 59.07 and 6.51 ng/ mL after consumption of artichoke leaf extract (107 mg) [30]. In addition, the half-lives of flavonoids in human plasma are short, usually in a range of a few hours [112]. In rats, after ad ministration of artichoke extract (107 mg/kg), the half-lives of total caffeic acid and luteolin were 3.08 and 2.50 h. These factors limit the capability of dietary flavonoids to act as antioxidant in plasma in vivo Chronic consumption of flavonoid-rich foods does not result in the sign ificant increase of am ounts of flavonoids in plasma. For example, the concentrations of querce tin at the steady-state in human plasma are less than 1 M [113].

PAGE 100

100 Besides the poor absorption, caffeolyquinic acids and flavonoids are highly metabolized in the intestine and liver. Flavonoids and caffeic acid are good substr ates of phase II enzymes and can be metabolized to glucuronidation, met hylation and sulfation [31, 96, 114, 115]. These biotransformations affect the physical propertie s of flavonoids, making them more water soluble and may affect their antioxidant activity. Fl avonoid metabolites generally are less potent antioxidants than their parent compounds because of the modification of their catechol and phenol group [47, 48, 116, 117]. Furthermore, the major part of ingest ed flavonoids is not absorbed and is largely degrad ed by the intestinal microflo ra [118]. The breakdown products may have antioxidant or non-an tioxidant activities [119, 120]. More over, our in vitro preliminary study demonstrated th at a plasma concentration higher than 1 g/mL of luteolin and quercetin was require d to increase the antioxidant activity in plasma above base line using the ORAC assay. Th erefore, it might be po ssible that the maximum plasma concentrations of luteolin and quercetin are below the plasma con centration necessary to expect an increase in the antioxidant activity. In conclusion, the in vitro antioxidant activity of artichoke and its compounds could not be confirmed in a rat model. This lack of effect might be due to the low bioavailabilty of caffeoylquinic acids and flavonoids Therefore, the pharmacokineti cs study of these compounds should be performed.

PAGE 101

101 Table 5-1. ORAC values of artichoke extract Sample mol TE/g artichoke extract mean SEM Artichoke extract 1623.36 2.84 Note: ORAC values are expressed as mi cromole of Trolox equivalent per gram.

PAGE 102

102 Table 5-2. Relative ORAC values of pure chemicals with antioxidant activity Compound Relative Trolox Equivalent mean SEM Caffeic acid 3.48 0.08 Dihydrocaffeic acid 2.83 0.06 Chlorogenic acid 1.83 0.22 Cynarin 6.73 0.06 Luteolin 5.16 0.05 Luteolin-7-O-glucoside 4.35 0.13 Quercetin 5.30 0.03 Note: ORAC values are expresse d as relative Trolox equivalent.

PAGE 103

103 Table 5-3. ORAC values of plasma samples Test samples Dose (mg/kg) ORAC (mmol trolox equivalent/L) mean SEM control 0.27 0.04 Artichoke 500 0.22 0.03 1000 0.21 0.04 Luteolin 25 0.38 0.05 50 0.31 0.03 Quercetin 25 0.31 0.05 Note: Rats were orally given ar tichoke extract, luteo lin and quercetin at the different doses indicated for 2 h. Data represent mean value SEM (n = 8).

PAGE 104

104 Table 5-4. ORAC values of plasma samples ORAC (mmol trolox equivalent/L) mean SEM Compounds Day 0 Day7 Day14 Day21 Control 0.44 0.03 0.50 0.070.43 0.05 0.40 0.03 Artichoke 500 mg/kg 0.34 0.04 0.35 0.030.37 0.03 0.33 0.04 Artichoke 1000 mg/kg 0.49 0.030.41 0.020.43 0.03 0.43 0.02 Luteolin 25 mg/kg 0.44 0.03 0.38 0.030.45 0.04 0.39 0.03 Luteolin 50 mg/kg 0.46 0.02 0.37 0.030.37 0.03 0.45 0.02 Quercetin 25 mg/kg 0.42 0.010.33 0.020.31 0.04 0.39 0.03 Note: Rats were orally given ar tichoke extract, luteo lin and quercetin at the different doses indicated for 21 days. Data represent mean value SEM. (n = 8).

PAGE 105

105 Table 5-5. Plasma urate concentrations in rats after administration of artichoke extract and phenolic compounds Uric acid levels (ug/ml) Treatment Dose (mg/kg) N Day 0 Day 7 Day 14 Day 21 Control -8 4.82 0.283.37 0.384.05 0.40 4.34 0.67 Artichoke 5008 3.37 0.573.04 0.313.11 0.18 3.68 0.53 10008 3.79 0.333.98 0.544.19 0.48 4.64 0.56 Luteolin 258 2.65 0.153.09 0.223.86 0.51 4.01 0.63 508 3.37 0.293.36 0.353.49 0.27 3.69 0.31 Quercetin 258 2.41 0.172.33 0.133.16 0.15 3.40 0.34 Note: Rats were orally given ar tichoke extract, luteo lin and quercetin at the different doses indicated for 21 days. Data represent mean value SEM (n = 8).

PAGE 106

106 Table 5-6. Plasma glutathione peroxidase activity in rats after administrati on of artichoke extract and phenolic compounds GPx Activity (nmol/min/ml) mean SEM Treatment Day 0Day 21 Control 5436 417.17279 513.5 Artichoke 500 mg/kg 5356 281.96970 249.8 Artichoke 1000 mg/kg 7263 271.67080 852.5 Luteolin 25 mg/kg 5501 166.46529 424.8 Luteolin 50 mg/kg 6282 454.25692 490.2 Quercetin 25 mg/kg 6418 460.76282 537.3 Note: Rats were orally given ar tichoke extract, luteo lin and quercetin at the different doses indicated for 21 days. Data represent mean value SEM. (n = 8).

PAGE 107

107 OH O H O OH OH O H O OH Caffeic acid Dihydrocaffeic acid OH O OH O HOOC O OH OH O O H O H OH OH O OH HOOC C O OH OH Cynarin Chlorogenic acid O O OH O H OH OH OH Quercetin R=H, Luteolin R=Glc,Luteolin-7-O-glucoside Figure 5-1. Structures of caffeic acid derivatives and flavonoids. O O OH OH OH RO

PAGE 108

108 CHAPTER 6 PHARMACOKINETICS OF LUTEOLIN AND ITS METABOLITES IN RATS Background Luteolin, one of the active components in artichoke leaves ( Cynara scolymus L.), had strong xanthine oxidase inhib itory and antioxidant activity in vitro as shown in our previous study. Luteolin also has been reported to be non-mutagenic [121], antitumorigenic [122], and has been recognized as an inhibitor of protein kinase C [123 ]. Therefore, in view of the potential of luteolin as a pharmacological agent, the pha rmacokinetics should be carefully studied. At present, the pharmacokinetics of luteolin has not been fully ch aracterized, although a number of studies have been re ported in animals and humans. These studies showed that the concentration of luteolin in plasma is low after oral administration. However high amount of metabolites, for example, luteolin conjugates we re found in systemic circulation [30, 104]. The bioavailability of luteolin is unknown. Therefore, to obtain more information about absorption and disposition, the pharmacokinetics of luteolin in rats treated with oral and intravenous administration of luteolin should be performed. Specific Aims Pharmacokinetic analysis of luteol in and its metabolites in rats. Materials and Methods Materials Luteolin (99%) was purchased from Indofine Chemical Company, Inc. (Somerville, NJ, USA). Naringenin ( 96%) (internal standard) was purchased from Roth Carl Roth GmbH+Co. (Germany). Acetone, acetonitrile (CH3CN), acetic acid, Dimethyl sulfoxide (DMSO), methanol, orthophosphoric acid (85% p.a.) were obtained from Fisher Scie ntific (Fair Lawn, NJ, USA). L (+)-ascorbic acid ( 99.9%) was obtained from Acros organics (New Jersey, USA).

PAGE 109

109 Trifluoroacetic acid was obtained from Fluka (Milwaukee, WI, USA). -glucuronidase/sulfatase (type HP-2, Helix pomatia), polyethylene glycol 200, and sodi um dihydrogenphosphate monohydrate (NaH2PO4.H2O), were obtained from Sigma Chem ical Company (St. Louis, MO, USA). All buffers and aqueous solutions were prepared with purified water obtained from a NANOPure system from Barnstead (Dubuque, IA, USA). Stock, Work Solutions, and Preparation of Calibration Standards The stock solutions of luteolin 10.0 mg/m L and naringenin (internal standard) 22.05 mg/mL were prepared in DMSO and kept at -80oC. Luteolin stock solution : (10 mg/mL) : Luteolin 20.0 mg was accurately weighed and transferred to a 2.0 mL volumetric flask. The st andard was then dissolv ed in DMSO and the volume was completed with the same solvent. Naringenin stock solution: 22.05 mg/mL : Naringenin 110.25 mg was weighed and transferred to a 5 mL volumetric flask and then dissolved in DMSO. The volume was completed with the same solvent. Luteolin work solution: 500 g/mL : Volume of 100 L from luteolin stock solution was accurately transferred to a 2 mL volumetric flask. The volume was completed with methanol and mixed thoroughly. The final concentration of luteolin was 500 g/mL. Naringenin work solution (internal standard): 1.05 mg/mL : Volume of 95.0 L from naringenin stock solution was transferred to a 2. 0 mL volumetric flask and then completed the volume with methanol. The final concen tration of naringenin was 1.05 mg/mL. Standard solutions of luteolin in plasma : From the luteolin work solution, five different concentrations of standard solutions and three qu ality controls (QC) were prepared in methanol. Then 10 L of standard solutions was added to 200 L plasma according Table 6-1.

PAGE 110

110 Standard solutions of luteolin in urine : From the luteolin stock solution and work solution, five different concentrations of standa rd solutions and three QC were prepared in methanol and 10 L of standard solutions was added to 200 L urine according Table 6-2. Animals and Experimental Protocols Animals Male Sprague-Dawley rats, weighing 250-350 g. were purchased from Harlan (IN, USA) and divided into the experimental groups; cont aining 11 rats per group. They were housed in plastic cages. They were allowed one week to adapt to their environment before used for experiments. All the animals were maintained on a 12hr/12hr light/dark cy cle. They were given standard chow and water ad libitum during the course of the study. All animal experiments were performed according to the policies and guideline s of the Institutional Animal Care and Use Committee (IACUC) of the University of Florid a, Gainesville, USA (N IH publication # 85-23). Methods The pharmacokinetic studies were carried ou t by the sparse sampling approach wherein blood samples were collected from 8-11 different rats. Luteolin was administered in two groups of rats (n = 8-11 in each group). Group one received a single i.v. dose of 50 mg/kg of luteolin via a tail vein. Group two received the same dose oral ly by gavage. Luteolin was dissolved in 30% DMSO and 70% PEG200. For luteolin analysis, plasma samples (500 L per blood sample) were collected from sublingual vein into heparini zed tubes at 3, 5, 10, 30 min, and 1, 2, 4, 6, 12, 24 h for i.v. injection and at 5, 10, 15, 30, 45 min, and 1, 2, 4, 6, 12, 24 h for oral administration. The blood collections were separated into two different days apart by one week of wash out period (5-6 blood collections per day per animal). Fo r luteolin conjugates, plasma samples (1000 L per blood sample) were collected at 5, 10, 30 min and 1, 2, 4, 6, 12, 24 h for i.v. and oral

PAGE 111

111 administration. The blood collections were separa ted into two different days apart by one week of wash out period (3 blood collections per day per animal). The variability of the weight of each animal on both periods was not higher than 20 %. Prior to blood collection, the rats were anaesthetized with halothane and the blood loss was replaced with an equal volume of normal saline. The blood sample was centrifuged for 15 min at 4,000 rpm at 4oC. The supernatant in aliquots of 200.0 L was transferred into tubes and 10.0 L of 0.58 M acetic acid was added to each aliquot for stabilization. The pl asma samples were stored at -80oC until analysis. Urine was collected over 24 h and an ali quot of 50.0 mL was mixed with 1g ascorbic acid as antioxidant and stored at -80oC until analysis. Analytical Methods The plasma concentrations of unchanged free and conjugated luteolin in rat plasma and urine were determined by the method published earli er with a slight modification [124] Plasma samples and urine samples were analyzed using a reverse-phase partiti on mode of HPLC with diode array detector. A Shimadzu VP series HPLC system (Kyoto, Japan) equipped with an SPD-M10Avp diode array detector was used for this work. A Lichrospher 100 RP-18 (5 m. Merck KgaA) was used for the separation of lute olin. The column temperature was kept at 25oC. The eluents were (A) 50 mM phosphate buffer (NaH2PO4, pH 2.1) and (B) CH3CN.The following solvent gradient was applied: 20% B (6 min) and 20-50% B (21 min).The gradient was followed by 10 min column flushing and post-run equilibration, respectively. Total run time was 40 min. The flow rate was 1 mL/min. 40 L of each sample was injected into the RP-HPLC system. Chromatograms were acquired at 330nm. For the determination of total luteolin, 10.0 L of internal standard (naringenin, 1.05 mg/ml), 10.0 L of 0.5% (m/v) ascorbic acid and 20.0 L of acetic acid (0.58 M) were added to

PAGE 112

112 200.0 L of plasma sample; follo wed by the addition of 12.0 L of -glucuronidase/sulfatase solution. The mixture was incubated at 37oC for 1 h. Protein was precipitated by adding 240 L of acetone. The mixture was vortexed for 1 mi n and centrifuged for 15 min at 4000 rpm at 4oC. Then the supernatant was tran sferred to tubes containing 4.0 L of 0.5% (m/v) ascorbic acid and 8.0 L of 1 M trifluoroacetic acid and evaporated to dryness in a vacuum centrifuge. The residue was reconstituted in 60 L of methanol: water (1 :1, v/v), centrifuged fo r 10 min at 13200 rpm, and 40 L was injected into HPLC. For the determination of unchanged luteolin in plasma, the sample was extracted in the same manner as described above without adding the enzyme. The concentrations in urine was measured using the same method as plasma, except urine samples were centrifuged for 15 min at 13200 rpm after adding 240.0 L of acetone and then 40.0 L of the supernatant was injected into HPLC. The conjugates (glucuronides or sulfates) of luteolin were calculated by subtracting total luteolin with unchanged luteolin. Data Analysis Plasma samples showed measurable concentrat ions for luteolin before administration of luteolin. Therefore, plasma concen trations at each time points were subtracted with baseline level before pharmacokinetic data analysis. Mean concen tration of luteolin and its conjugates versus time curves were generated in Grapad Prism (version 4.0, San Diego, CA). The pharmacokinetic parameters were determined by non-compartmental analysis and compartmental analysis using WinNonlin software package, version 3.1, (Pharsight Corporation, USA). The mean data was used for both analyses.

PAGE 113

113 Non-compartmental PK analysis: The PK parameters determined were the areas under the concentration time curve (AUC), maximum concentration in plasma (Cmax), time to reach Cmax (Tmax), the elimination rate constant (ke), the elimination half life (t1/2), the volume of distribution (Vd) and the clearance (CL). AUC 0 last was calculated using linear/log trapezoidal method from time zero to last sampling point equa l to or above the lower limit of quantification. AUC 0 was calculated as AUC 0 last + AUCextra, and AUCextra was determined as the calculated last concentration (Clast)/ke. Both Cmax and Tmax were obtained from the plots of plasma concentration versus time. The ke was obtained by linear regres sion of the terminal log linear phase of the concentration-time curve. The elimination half-life (t1/2) was determined as 0.693/ke. The volume of distribution of central compartment (V) was calculated as D/C0, where D is the dose. The clearance (Cl) was calculated as D/AUC. The systemic bioavailability (F %) was calculated as F % = (AUC p.o. Dose i.v. /AUC i.v. Dose p.o.) 100. Compartmental PK analysis: Luteolin concentrations s howed better fit in a two compartment body model compared with one co mpartment body model. The equation for two compartment model (Figure 6-1.) is as followed: C = A.et + B.et (i.v.) Where C is the concentration of drug in pl asma at time t; A and B are mathematical coefficient; is the distribution rate constant; is the elimination rate constant; and t is time. After i.v. administration, AUC 0was calculated using following equation: AUC 0i.v. = A/ + B/ The elimination half life was calculated as ln (2/Ke). The volume of distribution of central compartment (Vc) was calculated as Dose/Ke AUC. The volume of distribution of peripheral compartment (Vt) was calculated as Vc k12/k21. Th e clearance (Cl) was calculated as Dose/AUC.

PAGE 114

114 Goodness of fit was determined by the AIC (Aka ike Criteria) and SC (Schwartz Criteria). The lower the AIC and SC, the more appropriate the selected model. Statistical Analysis WinNonlin software package, version 3.1, (P harsight Corporation, USA) was used for statistical analysis. Data ar e given as mean with corre sponding standard deviation. Validation The method was validated over the range of con centration of luteolin present in plasma. The validation parameters of linearity, sensitivit y, specificity, precision, accuracy and stability were determined. The linearity of the calibration curves was de termined by least-squares linear regression method and expressed in terms of coefficient of determination (r2). The intraand inter-day precision and accuracy were measured by triplicate analyses of three different concentration levels (low, medium and high) of quality cont rol standards on the same day and on different days. The precision was based on the calculation coefficient of variation (CV %), and the accuracy was defined as the percent difference be tween the theoretical and measured values. The calibration was considered suitable if not more than 1/3 of the qua lity control standards showed a deviation from the theoretical values equal or gr eater than 15%, except at the lower limit of quantification (LLOQ), where it should not exceed 20%. Results Validation of Analytical Method to Measure Luteolin in Rat Plasma Linearity Calibration curve (n = 9) operating in the range of 100-10000 ng/mL fo r luteolin in rat plasma was linear (r2 > 0.99) (Figure 6-2).

PAGE 115

115 Sensitivity In this study, the limit of quantification (LLOQ) is defined as the lowest concentration for quality control. This concentration would be acceptable with the precision (%CV < 20), and accuracy (%error < 20).The LLOQ of lu teolin in plasma was 100 ng/mL. Specificity The methods provided good reso lutions between luteolin, -glucuronidase and interference in plasma and there was no endogenous interferen ce from plasma (Figure 6-3) in this assay, indicating the specific ity of this method. Precision, accuracy and recovery The precisions intraand inter-day for luteolin were satisfactory with CV values between 1.3 and 12.3%. Similarly, the accuracy of the assay obtained with quality control samples containing 300, 800, and 3000 ng/mL luteolin was between 94.2 and 106.3 % of the nominal values. The mean recovery assessed at three distinct levels of concentration (100, 500 and 10000 ng/mL) ranged from 95.7 to 106.4 % of the expect ed values. The results are summarized in Table 6-3. Stability Luteolin was stable under the tested conditions. The mean % remainings in rat plasma after 2 hours at room temperature were 98.78 5.34, 96.29 1.47, 102.4 7.76 for the low, medium and high concentrations, respectiv ely. The mean % remainings of luteolin after an evaporation and keep at -20 C for 24 hours were 106.68 8.97, 102.40 3.11 and 100.07 0.50 for the low, medium and high concentrat ions, respectively. Luteolin was stable on autosampler at 18 oC within 48 hours (Table 6-4).

PAGE 116

116 Validation of Analytical Method to Measure Luteolin in Rat Urine Linearity Calibration curve (n = 9) operating in the range of 500-50000 ng/mL fo r luteolin in rat urine was linear (r2 > 0.99) (Figure 6-4). Sensitivity The limit of quantification (LLOQ) of luteolin in urine was 500 ng/mL. Specificity Good resolutions be tween luteolin, -glucuronidase and interference in urine and no endogenous interference from urine (Figure 6-5) indicated the specificity of this method. Precision, accuracy and recovery The precisions intraand inter-day for luteolin were satisfactory with CV values between 0.30 and 13.25%. Similarly, the accuracy of the assay obtained with quality control samples containing 500, 3000, and 10000 ng/mL luteolin was between 98.21 and 109.28 % of the nominal values. The mean recovery assessed at three distinct levels of concentration (500, 3000 and 10000 ng/mL) ranged from 99.56 to 112.23 % of the expected values. The results are summarized in Table 6-5. Stability Luteolin was stable under the tested conditions. The mean % re mainings in rat urine after 2 hours at room temperature were 98.78 5.34, 96.29 1.47, 102.4 7.76 for the low, medium and high concentrations, respectively. Lute olin was stable on autosampler at 18 oC within 48 hours (Table 6-6).

PAGE 117

117 Pharmacokinetic Study of Luteolin Non-compartmental analysis Plasma levels of luteolin after oral and i.v. administration of luteolin: The concentration-time profiles and the pharmacokinetic parameters of luteolin after oral and i.v. administration are presented on Figure 6-6 a nd Table 6-7. For oral administration, plasma concentrations of luteolin attained maximum level of 5.49 g/ml at 0.08 h and decreased to below LOQ (100 ng/ml) after 1 h. Ke could not be calculated because the elimination phase was below LOQ. Our assumption was Ke after oral administration was similar to ke after i.v injection. Theref ore, the AUC 0p.o. was calculated using ke from i.v. The low bioavailability (F) of luteolin, 4.10 % at dose 50 mg/kg are presumably due to the significant first pass effect. For i.v. administration, the maximum concentration of luteolin was 23.42 g/mL at 0 h. The plasma concentration versus time profile of luteolin was biphasic, subdivided in to a distribution phase and a slow elimination phase for oral and intravenous administration. Plasma levels of luteolin conjugates aft er oral and i.v. administ ration of luteolin: The concentration-time profiles and the pharmacokinetic parameters of luteolin conjugates after oral and i.v. administration are pres ented on Figure 6-6 and Table 68. Plasma concentration of luteolin conjugates after oral and i.v administration of lute olin attained maximum level of 5.77 g/mL at 0.25 h and 4.31 g/ml at 0.08 h, respectively, and d ecreased to below LOQ at 24 h. The double peaks were found in luteolin conjugate s after oral and i.v. ad ministration at 0.25 and 1 h, respectively, suggesting it mi ght pass enterohepa tic circulation. Urinary excretion of luteolin and its meta bolites after oral and i.v. administration: Urinary excretion of luteolin a nd luteolin conjugates within 24 h after oral and intravenous

PAGE 118

118 administration of luteolin were very low ( 0.98 4.97% of the dose), suggesting these compounds are not primarily excreted via the urine (Table 6-10). Compartmental Analysis Figure 6-7 shows the fitted lu teolin concentrations versus time profiles with the two compartment body model. It can be seen that the model describes luteolin data very well. The AIC and SC were -11.18 and -9.59, representi ng a good fit. The PK parameters obtained by fitting the mean concentra tion versus time profiles of luteolin concentrations after i.v. treatment are presented in Table 6-9. Discussion and Conclusion Luteolin and luteolin conjugates were presented in rat plasma and urine after oral and intravenous administration. However, the conjugat es (glucuronides or su lfates) could not be further identified in this study. The analytical methods were developed for the parent compound, and the conjugates presumably coeluted with matrix compounds. The present of free luteolin suggested that some luteolin can escape the intestinal and he patic conjugation. Pharmacokinetic profiles of luteolin and lute olin conjugates in rat plasma are shown in Figure 6-6. When rats were given luteolin ( 50 mg/kg) in 30% DMSO: 70% PEG 200 orally, the maximum concentration of luteolin a nd luteolin conjugates were 5.48 and 5.77 g/mL at 5 min and 15 min, respectively. The total concentration of luteolin in ra t plasma at 5 min after dosing was 9.25 g/mL. Shimoi et al. [104] observed 15.5 3.8 nmol/mL ( 4.4 1.09 g/mL) of total luteolin concentration in rat plasma 30 min after administration of one single dose of luteolin (50 mol/kg, 14.3 mg/kg) in propylene glycol. In dog, the maximum concentration of luteolin was about 450 ng/mL at 3 h afte r a single oral dosing of Chrysanthemum morifolium Ramat extracts (102 mg/kg containi ng 7.60% luteolin, 7.75 mg/kg luteolin) [125]. In human, peak plasma

PAGE 119

119 concentrations of total luteolin were r eached within 0.5 h with maximum level of 156.5 92.29 ng/mL after a single oral dose of artichoke leaf extracts (153.8 mg c ontaining luteolin-7-Oglucosides; equivalent to 35.2 mg luteolin) [30] The differences observed between these studies could be explained by the differe nt initial dose administration of flavonoids and the different source of intake flavonoids. The rapid absorption of flavonoids has been repo rted in previous literature. When diosmin was administered to humans, a peak occurring 2 h after administration [126], whereas diosmetin administered per os to rats appeared in bl ood after 6 h as unchanged and glucuronated compound [127]. In pigs, after an oral dose of 50 mg/ kg, only 17 % of the quercetin administered was recovered in blood as free conjuga te and derivative products with in 8 h postadministration [128]. In humans, the peak in blood occurred more rapidly, approximately 2.9 h after the flavonol administration [103]. In rats, fla vonoids seemed to occur more rapidly. Luteolin given via gastric intubation, appeared in plasma af ter 15 min [104]. Apigenin given to rats via the intraperitoneal pathway appeared in plasma 30 min after admi nistration [105]. From these literatures, the presence of flavonoids in blood occurs within a few minutes to a few hours which are similar to our result. The low bioavailability of luteolin (F = 4.1 %) and high metabolite c oncentrations indicate first pass metabolism. Absorbed luteolin co uld undergo biotransformation (methylation, glucuronidation or sulfation) as shown in previous literature. In vitro experiments demonstrated that 74 % of luteolin was conjugated to gluc uronic acid after incubation with microsomal samples from human intestine. Most common bi nding sites of the mol ecule were the hydroxyl groups in the 3 and 4 position (51% and 44%) [96]. Bo ersma et al. [96] found three glucuronosyl conjugates of luteolin, the 7-O-, the 3 -Oand the 4 -O-glucuronosyl luteolin after

PAGE 120

120 incubation with intestine microsom es and liver microsomes from rat and man. Shimoi et al. [104] investigated the absorpti on of luteolin by rat everted small intestine. Luteolin was recovered in rat plasma as two metabolites, glucuronidate or sulfate forms of O-methylate conjugate. Only a small part of the compound remained unconjugated. Murota et al. [129] reported the uptake and transport of flavonoids aglycones by human inte stinal Caco-2 cells. Th e flavonoids, quercetin, kaempferol, luteolin and apigeni n, were converted to their gluc uronide/sulfates by Caco-2 cells, and the level of the intact aglycone form was le ss than those of the glucuronide/sulfates in the basolateral solution. To our knowledg e, this was the first study on bioavailability of luteolin, thus a comparable data is lacking. However, the bioava ilability of luteolin is similar to that of quercetin. Chen et al. [95] reported the system ic bioavailability of quercetin and quercetin conjugates as 5.3% and 55.8%, resp ectively in rats. Moreover af ter oral administration of quercetin, about 93.3% of quercetin was metabolized in the gut, with only 3.1% metabolized in the liver. Only small amounts of luteolin and luteolin conjugates were found to be eliminated in the urine in our study. This is consis tent with the observations by others. Shimoi et al. [104] found excretory recovery for 24 h as unmodified luteolin from the urine was about 4 % in rats. Luteolin conjugates was recovered only 1.99 1.50 % after intake of luteolin-7O -glucoside (equivalent to 35.2 mg luteolin) [30]. Only 0.58 % of apigenin was recovered in urine samples within 24 h after parsley ingestion in huma n [130]. Gugler et al. [131] found that after intravenous administration of a 100 mg quercetin, only 0.65 % of the dose was recovered in the form of unchanged quercetin, while 7.4 % of the dose was excr eted in the urine in the form of conjugated metabolite of quercetin. In a phase I clinical tria l of quercetin (in fifty-one cancer patients) at

PAGE 121

121 dose of 60 to 2000 mg/m2, the percentage of quercetin in urine over 24 h ranged from 0.03% to 7.6% [132].Therefore, urinary elimination of luteo lin is not the main ex cretion route in rats. Our study found multiple peaks in a plasma concentration-time profile of luteolin conjugates after oral and intravenous administra tion of luteolin suggesting an enterohepatic recirculation of luteolin which is similar to other studies. Liu et al. [39] reported a rapidly absorbed and rapidly metabolized of aglycones such as apigenin and quercetin into phase II conjugates, which were then excreted back into the lumen; following and enteric and enterohepatic recycling. Ma et al [133] reported an enterohepatic recirculation of naringenin in rat plasma. Our data did not show multiple peaks in a plasma concentration-time profile of free luteolin after intravenous and oral administration presumably due to the limit of data time points. In the present investigation, pharmacokinetics of luteolin and its metabolites in rats were studied. After oral administration of luteolin, lu teolin was rapidly absorbed and metabolized in plasma; moreover, plasma-concentration-time curves of luteolin metabolit es revealed secondary peaks. The bioavailability of luteolin is low and the urinary excretion of luteolin and its conjugates did not dominate. This study could explain a lack of in vivo activity of artichoke leaf and its compounds on xanthine oxidase inhibitory and an tioxidant activity Moreover, it can be used to predict the in vivo activity of other herbal products that contain this compound.

PAGE 122

122 Table 6-1. Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in plasma Standard Luteolin in methanol ( g/mL) Luteolin in plasma (ng/mL) 1 10.50500 2 21.001000 3 105.005000 4 210.0010000 5 1050.0050000 QC1 10.50500 QC2 63.003000 QC3 210.0010000

PAGE 123

123 Table 6-2. Concentrations of standard solutions used for the calibration curves and quality controls (QCs) of luteolin in urine Standard Luteolin in methanol ( g/mL) Luteolin in plasma (ng/mL) 1 2.10100 2 10.50500 3 21.001000 4 105.005000 5 210.0010000 QC1 2.10100 QC2 16.80800 QC3 63.003000

PAGE 124

124 Table 6-3. Intra-day (n = 3), interday (n = 9), and recovery (n = 3) assay parameters of luteolin in rat plasma. Precision expressed as CV %, accuracy and recovery as % of the theoretical concentration Intra-day QC1 100 ng/mL QC2 800 ng/mL QC3 3000 ng/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 3.82 12.34 8.711.335.705.291.73 5.92 2.28 Accuracy 94.20 98.72 100.21102.57104.06104.30106.25 104.68 104.12 Inter-day QC1 100 ng/mL QC2 800 ng/mL QC3 3000 ng/mL Precision 9.65 2.823.02 Accuracy 98.17 104.69104.21 Recovery Luteolin-100 ng/mL Luteo lin-500 ng/mL Luteolin-10000 ng/mL % 95.78 96.42106.40 CV% 10.94 8.465.48

PAGE 125

125 Table 6-4. The stability test af ter 48 hours on autosampler at 18oC. Data represents the percentage remaining of luteolin in plasma SD % Remaining on autosampler Luteolin concentration 12 hours24 hours48 hours Low-100 ng/mL 108.81 15.1390.83 11.3791.28 14.38 Medium-500 ng/mL 100.10 2.4498.34 2.98114.21 13.10 High-10000 ng/mL 99.70 0.6699.89 2.40100.52 1.33

PAGE 126

126 Table 6-5. Intra-day (n = 3), interday (n = 9), and recovery (n = 3) assay parameters of luteolin in rat urine. Precision expressed as CV %, accuracy and recovery as % of the theoretical concentration Intra-day QC1 500 ng/mL QC2 3000 ng/mL QC3 10000 ng/mL Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Precision 6.48 7.24 0.810.300.731.522.96 0.61 1.66 Accuracy 98.21 100.12 98.51101.81101.92100.98110.01 112.45 108.99 Inter-day QC1 500 ng/mL QC2 3000 ng/mL QC3 10000 ng/mL Precision 13.25 3.241.37 Accuracy 98.72 103.49109.28 Recovery Luteolin-500 ng/mL Luteo lin-3000 ng/mL Luteolin-10000 ng/mL % 99.56 108.46112.23 CV% 3.19 3.323.21

PAGE 127

127 Table 6-6. The stability test of luteolin in urine after 48 hours on autosampler at 18oC. Data represents the percentage remaining of luteolin SD % Remaining on autosampler Luteolin concentration 24 hours48 hours Low-500 ng/mL 97.44 2.3892.98 0.61 Medium-3000 ng/mL 98.77 0.7198.85 1.48 High-10000 ng/mL 100.10 0.53100.32 1.65

PAGE 128

128 Table 6-7. Pharmacokinetic parameters of luteolin after oral and iv admini stration of luteolin at dose 50 mg/kg Parameter Luteolin oralLuteolin iv Tmax (h) 0.080 Cmax ( g/mL) 5.4823.42 Ke (1/h) ND0.08 t (h) ND8.94 Cl/F (L/h/kg) ND Vd/F (L/kg) ND Cl (L/h/kg) 2.14 Vd (L/kg) 27.58 AUC0-last (h* g/mL) 0.8720.55 AUC0(h* g/mL) 0.9623.39 F (%) 4.10 Note: All Pk parameters are mean values calculated by a normalized dose (50 mg/kg)

PAGE 129

129 Table 6-8. Pharmacokinetic parameters of luteolin conjugates after oral and iv administration of luteolin at dose 50 mg/kg Parameter Luteolin conjugates oralLuteolin conjugates iv Tmax (h) 0.250.08 Cmax ( g/mL) 5.774.31 Ke (1/h) 0.100.14 t (h) 6.574.98 AUC0-last (h* g/mL) 11.4912.83 AUC0(h* g/mL) 15.6815.26 Note: All pk parameters are mean values calculated by a normalized dose (50 mg/kg)

PAGE 130

130 Table 6-9. Pharmacokinetic parameters of luteolin after i.v. administratio n of luteolin 50 mg/kg. Data was fitted to a two-compartment model. Parameter Luteolin i.v. A ( g/mL) 9.66 1.14 B ( g/mL) 1.36 0.16 (1/h) 1.95 0.32 (1/h) 0.08 0.01 K12 (1/h) 1.24 0.25 K21 (1/h) 0.31 0.06 Ke (1/h) 0.48 0.06 Vc (L/kg) 4.54 0.48 Vt (L/kg) 18.26 2.24 Cl (L/h/kg) 2.18 0.13 t1/2 (h) 0.36 0.06 t1/2 (h) 9.15 1.15 t1/2 Ke (h) 1.44 0.17 AUC (h g /mL) 22.93 1.39 Cmax ( g /mL) 11.02 1.15 Note: All pk parameters are mean S.D.

PAGE 131

131 Table 6-10. The excretory recovery for 24 h of luteol in and luteolin conjugat es in urine after oral and i.v administration of luteolin at dose 50 mg/kg Treatment % Luteolin% Luteolin conjugates oral 0.98 0.983.91 0.52 i.v. 2.05 0.904.97 1.68 Note: Data expressed as mean SD (n = 11)

PAGE 132

132 bolus IV 1 2 K12K21Ke Figure 6-1. Two-compartment models after Intravenous injection. 1 is the central compartment, 2 is the peripheral compartment, Ke is the first order elimination rate constant, K12 is the rate constant for transfer of drug fro m the central compartment to the peripheral compartment and K21 is the rate constant for transfer of drug from the peripheral compartment to the central compartment.

PAGE 133

133 0 2500 5000 7500 10000 12500 0.0 0.5 1.0 1.5 2.0 2.5Luteolin [ng/ml]Ratio( area luteolin: area IS) Figure 6-2. Mean calibration curves (n = 9) of luteolin in plasma Vertical bars represent the standard deviations (SD) of the means. Y = 0.0002057 X 0.003217 R = 0.9978

PAGE 134

134 Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 mVolts 0 20 40 60 80 100 120 140 160 180 200 Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 mVolts 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 Figure 6-3. The HPLC chromatogram of luteolin and naringenin (IS) in plasma. A) With out glucuronidase. B) With -glucuronidase/sulfatase. A B N arin g eninLuteolin Luteolin N arin g enin

PAGE 135

135 0 10000 20000 30000 40000 50000 60000 0.0 2.5 5.0 7.5 10.0 12.5Luteolin [ng/mL]Ratio (Area Luteolin:Area IS) Figure 6-4. Mean calibration curves (n = 9) of luteolin in urine. Vertical bars represent the standard deviations (SD) of the means. Y = 0.0002X 0.0703 R 2 = 0.9992

PAGE 136

136 Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 mVolts-10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Minutes0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 mVolts 0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 Figure 6-5. The HPLC chromatogram of luteolin and naringenin (IS) in urine. A) With out glucuronidase/ sulfatse. B) With -glucuronidase/ sulfatase. A B N arin g eninLuteolin N arin g eninLuteolin

PAGE 137

137 A 0 5 10 15 20 25 30 0.01 0.1 1 10 100Luteolin p.o. Luteolin i.v. Time(h)log [luteolin](ug/mL plasma) B 0.0 2.5 5.0 7.5 10.0 12.5 15.0 0.01 0.1 1 10 100Luteolin conjugates i.v. Luteolin conjugates p.o. Time(h)Log [Luteolin] (mg/ml) Figure 6-6. Plasma concentration-time curves. A) Luteolin. B) Luteolin conjugates. After oral and intravenous administration of 50 mg/kg to ra ts (n = 8-11). Error bars refers to the standard deviation of concentration data at each sampling time point.

PAGE 138

138 Figure 6-7. Fitted luteolin concen trations after i.v. in jection. Experimental points represent the means of 8-11 rats. 0.1 1.0 10.0 100.0 0 5 10 15 20 25 Time (h) Observed Predicted

PAGE 139

139 CHAPTER 7 CONCLUSION Gout is a common metabolic di sorder in human. It results from deposits of needle-like crystals of uric acid in connec tive tissue, in the join t space between two bones, or in both. These depositions lead to inflammatory arthritis, which causes swelling, redness, heat, pain, and stiffness in the joints. The common treatments fo r an acute attack of gout are colchicine, nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids. Allopurinol, a xanthine oxidase inhibitor, is used for the prev ention of chronic gout attacks. Its use is limited by unwanted side effects such as hypersensitivity problems. Therefore, alternatives are required. Leaf of Artichoke ( Cynara scolymus L ) is a good source of polyphenolic compounds such as monoand dicaffeoylquinic ac ids and flavonoids. Polyphenolic compounds have a role in the prevention of degenerative diseases such as cancer, cardiovascular disease and neurodegenerative diseases, whic h is usually linked to two prope rties: antioxidant activity and inhibition of certain enzymes such as xanthine oxidase. Therefore, artichoke leaves containing polyphenolic compounds may show xanthine oxidase inhibitory activity and antioxidant activity. In this study, artichoke leaf extract a nd caffeoylquinic acids showed weak or no XO inhibitory activity in vitro ; whereas, the inhibitions of most flavonoids on XO were stronger than a standard compound, allopurinol. However, after oral and intraperitoneal administration of different doses of artichoke and polyphenolic compounds in rats, none of the test compounds could decrease serum urate levels. This resu lt of the XO study was similar to that of the antioxidant study. The study of antio xidant activity of artichoke a nd its components also showed that although there was an antioxidant activity in vitro the antioxidant activity in vivo was not found after oral treatment. This l ack of XO and antioxidant activity in vivo might be explained by low absorption, high first pass effect through gut, in testine and liver, rapid excretion into

PAGE 140

140 urine and bile, degradation and metabolization by the colonic microflora. In addition, the metabolites may differ from the native substances in terms of biological activity. Therefore, the further studies of bioavailability of polyphenol ic compounds and the activ ity of metabolites are essential. The activity of metabolites such as lute olin-7-O-glucuronide has shown a weaker inhibition on XO comparing to luteolin in vitro Moreover the pharmacokinetics of luteolin showed that luteolin has low bi oavailability after oral administ ration. These results could explain a lack of activity of artichoke leaf extract and its components on xanthine oxidase inhibitory activity and antioxidant activity in vivo Therefore, we can conclude artichoke leaf does not seem to be an alternative for treating gout.

PAGE 141

141 LIST OF REFERENCES 1. Pittler, M.H., C.O. Thompson, and E. Ernst, Artichoke leaf extract for treating hypercholesterolaemia. Cochrane Database Syst Rev, 2002(3): p. CD003335. 2. Thompson Coon, J.S. and E. Ernst, Herbs for serum cholesterol reduction: a systematic view. J Fam Pract, 2003. 52 (6): p. 468-78. 3. Englisch, W., et al., Efficacy of Artichoke dry extract in patients with hyperlipoproteinemia. Arzneimittelforschung, 2000. 50 (3): p. 260-5. 4. Adzet, T., J. Camarasa, and J.C. Laguna, Hepatoprotective activity of polyphenolic compounds from Cynara scolymus against CCl4 toxicity in isolated rat hepatocytes. J Nat Prod, 1987. 50 (4): p. 612-7. 5. Clifford, M., Chlorogenic acids and other cinnamatesnature, occurence, dietary burden, absorption and metabolism. J.Sci.Food Agric., 2000. 80 : p. 1033-1043. 6. Gebhardt, R., Antioxidative and protective properties of extracts from leaves of the artichoke (Cynara scolymus L.) against hy droperoxide-induced oxidative stress in cultured rat hepatocytes. Toxicol Appl Pharmacol, 1997. 144 (2): p. 279-86. 7. Zapolska-Downar, D., et al., Protective properties of ar tichoke (Cynara scolymus) against oxidative stress i nduced in cultured endothe lial cells and monocytes. Life Sci, 2002. 71 (24): p. 2897-08. 8. Jimenez-Escrig, A., et al., In vitro antioxidant activities of edible artichoke (Cynara scolymus L.) and effect on biom arkers of antioxi dants in rats. J Agric Food Chem, 2003. 51 (18): p. 5540-5. 9. Brown, J.E. and C.A. Rice-Evans, Luteolin-rich artichoke extr act protects low density lipoprotein from oxidation in vitro. Free Radic Res, 1998. 29 (3): p. 247-55. 10. Kraft, K., Artichoke leaf extract-recent findings reflecting effects on lipid metabolism, liver and gastrointestinal tratcs. Phytomedicine, 1997: p. 367-378. 11. Saenz Rodriguez, T., D. Garcia Gime nez, and R. de la Puerta Vazquez, Choleretic activity and biliary eliminati on of lipids and bile acids i nduced by an artichoke leaf extract in rats. Phytomedicine, 2002. 9 (8): p. 687-93. 12. Gebhardt, R., Artischockenextrakt in vitro Na chweis einer Hemmwirkung auf die Cholesterinbiosynthese. Med Welt, 1995. 46 : p. 348-350. 13. Gebhardt, R., Neue Erkenntnisse zur Wirkung vo n Artischockenblaetterextrakt]. Z Allg Med, 1996. 72 : p. 20-2.

PAGE 142

142 14. Fintelmann, V., Therapeutic profile and mechanism of action of artichoke leaf extract: hypolpemic, antioxidant, hepatoprotec tive and choleretic properties. Phytomedicine, 1996. Supplement 1:50 15. Gebhardt, R., Inhibition of hepatic cholesterol biosynth esis by artichoke leaf extracts is mainly due to luteolin. Cell Bio Toxicol, 1997(13): p. 58. 16. Perez-Garcia, F., T. Adzet, and S. Canigueral, Activity of artichoke leaf extract on reactive oxygen species in human leukocytes. Free Radic Res, 2000. 33 (5): p. 661-5. 17. Maros T.e.a., Wirkungen der Cynara scolymus Ex trakte auf die regeneration der Rattenleber. Arzneimittelforschung, 1966. 16 : p. 127-129. 18. Holtmann, G., et al., Efficacy of artichoke leaf extract in the treatment of patients with functional dyspepsia: a sixweek placebo-controlled, doubl e-blind, multicentre trial. Aliment Pharmacol Ther, 2003. 18 (11-12): p. 1099-105. 19. Bundy, R., et al., Artichoke leaf extract reduces symp toms of irritable bowel syndrome and improves quality of life in otherwise he althy volunteers sufferi ng from concomitant dyspepsia: a subset analysis. J Altern Complement Med, 2004. 10 (4): p. 667-9. 20. Simon Mills, k.B., Principles and Practice of Phytothe rapy ( Modern Herbal Medicine) 2000: Churchill Livingstone. 533-438. 21. Zhu, X., H. Zhang, and R. Lo, Phenolic compounds from the leaf extract of artichoke (Cynara scolymus L.) and th eir antimicrobial activities. J Agric Food Chem, 2004. 52 (24): p. 7272-8. 22. Schutz, K., et al., Identification and quantification of caffeoylquinic acids and flavonoids from artichoke (Cynara scolymus L.) heads, juice, and pomace by HPLC-DADESI/MS(n). J Agric Food Chem, 2004. 52 (13): p. 4090-6. 23. Mulinacci, N., et al., Commercial and laboratory extra cts from artichoke leaves: estimation of caffeoyl esters and flavonoidic compounds content. J Pharm Biomed Anal, 2004. 34 (2): p. 349-57. 24. Wang, M., et al., Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.). J Agric Food Chem, 2003. 51 (3): p. 601-8. 25. Joanne Barnes, L.A.a.a.J.D.P., Herbal medicines: A guide for healthcare professionals 2 ed. 2002: The pharmaceutical Press. 26. Plumb, G.W., Garca-Conesa, M.T., roon, P.A ., Rhodes, M., Ridley, S., Williamson, G., Metabolism of chlorogenic acid by human plasma, liver, liver, intestine and gut microflora. J. Sci. Food Agric., 1999. 79 : p. 390-392.

PAGE 143

143 27. Andreasen, M.F., et al., Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals. J Agric Food Chem, 2001. 49 (11): p. 5679-84. 28. Takenaka, M., T. Nagata, and M. Yoshida, Stability and bioavailabili ty of antioxidants in garland (Chrysanthemum coronarium L.). Biosci Biotechno l Biochem, 2000. 64 (12): p. 2689-91. 29. Olthof, M.R., P.C. Hollman, and M.B. Katan, Chlorogenic acid and caffeic acid are absorbed in humans. J Nutr, 2001. 131 (1): p. 66-71. 30. Wittemer, S.M., et al., Bioavailability and pharmacokinetic s of caffeoylquinic acids and flavonoids after oral administration of Artichoke leaf extracts in humans. Phytomedicine, 2005. 12 (1-2): p. 28-38. 31. Moridani, M.Y., H. Sc obie, and P.J. O'Brien, Metabolism of caffeic acid by isolated rat hepatocytes and subcellular fractions. Toxicol Lett, 2002. 133 (2-3): p. 141-51. 32. Kuhnau, J., The flavonoids. A class of semi-essen tial food components: their role in human nutrition. World Rev Nutr Diet, 1976. 24 : p. 117-91. 33. Kim, D.H., et al., Intestinal bacterial metabolism of flavonoids and its relation to some biological activities. Arch Pharm Res, 1998. 21 (1): p. 17-23. 34. Winter, J., et al., C-ring cleavage of flavonoids by human intestinal bacteria. Appl Environ Microbiol, 1989. 55 (5): p. 1203-8. 35. Macdonald, I.A., J.A. Mader, and R.G. Bussard, The role of rutin and quercitrin in stimulating flavonol glycosidase activity by cu ltured cell-free microbi al preparations of human feces and saliva. Mutat Res, 1983. 122 (2): p. 95-102. 36. Nemeth, K., et al., Deglycosylation by small intestinal epithelial cell beta-glucosidases is a critical step in the absorption and met abolism of dietary flav onoid glycosides in humans. Eur J Nutr, 2003. 42 (1): p. 29-42. 37. Day, A.J., et al., Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactas e phlorizin hydrolase. FEBS Lett, 2000. 468 (2-3): p. 166-70. 38. Arts, I.C., A.L. Sesink, and P.C. Hollman, Quercetin-3-glucoside is transported by the glucose carrier SGLT1 across the brush bor der membrane of rat small intestine. J Nutr, 2002. 132 (9): p. 2823; author reply 2824. 39. Liu, Y., et al., Enteric disposition and recycling of flavonoids and ginkgo flavonoids. J Altern Complement Med, 2003. 9 (5): p. 631-40. 40. Cotelle, N., et al., Antioxidant properties of hydroxy-flavones. Free Radic Biol Med, 1996. 20 (1): p. 35-43.

PAGE 144

144 41. Scalbert, A., et al., Dietary polyphenols and the prevention of diseases. Crit Rev Food Sci Nutr, 2005. 45 (4): p. 287-306. 42. Halliwell, B., Gutteridge, J.M.C., In free radicals in bilogy and medicine 2 ed. 1989: Clarendron Press. 422-437. 43. McAnlis, G.T., et al., The effect of various dietary flav onoids on the susceptibility of low density lipoproteins to oxidation in vitro us ing both metallic a nd non-metallic oxidising agents. Biochem Soc Trans, 1997. 25 (1): p. 142S. 44. Fiander, H. and H. Schneider, Dietary ortho phenols t hat induce glutathione Stransferase and increase the resistance of cells to hydrogen peroxide are potential cancer chemopreventives that act by two mechanisms: the alleviation of oxidative stress and the detoxification of mu tagenic xenobiotics. Cancer Lett, 2000. 156 (2): p. 117-24. 45. Noroozi, M., W.J. Angerson, and M.E. Lean, Effects of flavonoids and vitamin C on oxidative DNA damage to human lymphocytes. Am J Clin Nutr, 1998. 67 (6): p. 1210-8. 46. Terao, J., Dietary flavonoids as antioxidants in vivo: conjugated metabolites of (-)epicatechin and quercetin participate in antioxidative defense in blood plasma. J Med Invest, 1999. 46 (3-4): p. 159-68. 47. Morand, C., et al., Plasma metabolites of quercetin and their antioxidant properties. Am J Physiol, 1998. 275 (1 Pt 2): p. R212-9. 48. Janisch, K.M., et al., Properties of quercetin conjugates : modulation of LDL oxidation and binding to human serum albumin. Free Radic Res, 2004. 38 (8): p. 877-84. 49. Arai, Y., et al., Dietary intakes of flavonols, flavon es and isoflavones by Japanese women and the inverse correlation between quercet in intake and plasma LDL cholesterol concentration. J Nutr, 2000. 130 (9): p. 2243-50. 50. Akaike, T., et al., Dependence on O2generation by xant hine oxidase of pathogenesis of influenza virus infection in mice. J Clin Invest, 1990. 85 (3): p. 739-45. 51. Ferrandiz, M.L. and M.J. Alcaraz, Anti-inflammatory activity and inhibition of arachidonic acid metabolism by flavonoids. Agents Actions, 1991. 32 (3-4): p. 283-8. 52. Kokoglu, E., et al., Xanthine oxidase levels in human brain tumors. Cancer Lett, 1990. 50 (3): p. 179-81. 53. McCord, J.M., Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med, 1985. 312 (3): p. 159-63. 54. Nishino, T., et al., Conversion of xanthine dehydrogenas e into oxidase and its role in reperfusion injury. Biochem Soc Trans, 1997. 25 (3): p. 783-6.

PAGE 145

145 55. Parks, D.A. and D.N. Granger, Xanthine oxidase: bioc hemistry, distribution and physiology. Acta Physiol Scand Suppl, 1986. 548 : p. 87-99. 56. F. Borges, E.F., and F. Roleira, Progress towards the discovery of xanthine oxidase inhibitors. Current Medicinal Chemistry, 2002. 9 : p. 195-217. 57. Hille, R. and T. Nishino, Flavoprotein structure and mec hanism. 4. Xanthine oxidase and xanthine dehydrogenase. Faseb J, 1995. 9 (11): p. 995-1003. 58. Hille, R. and V. Massey, Tight binding inhibitors of xanthine oxidase. Pharmacol Ther, 1981. 14 (2): p. 249-63. 59. Hawkes, T.R., G.N. George, and R.C. Bray, The structure of the inhibitory complex of alloxanthine (1H-pyrazolo[3,4-d]pyrimidine -4,6-diol) with the molybdenum centre of xanthine oxidase from electronparamagnetic-resonance spectroscopy. Biochem J, 1984. 218 (3): p. 961-8. 60. Skibo, E.B., Noncompetitive and irreversible inhibition of xanthine oxidase by benzimidazole analogues acting at the functi onal flavin adenine dinucleotide cofactor. Biochemistry, 1986. 25 (15): p. 4189-94. 61. Rundles, R.W., E.N. Metz, and H.R. Silberman, Allopurinol in the treatment of gout. Ann Intern Med, 1966. 64 (2): p. 229-58. 62. Klinenberg, J.R., S.E. Goldfinger, and J.E. Seegmiller, The Effectiveness of the Xanthine Oxidase Inhibitor Allopurinol in the Treatment of Gout. Ann Intern Med, 1965. 62 : p. 639-47. 63. Young, J.L., Jr., R.B. Boswell, and A.S. Nies, Severe allopurinol hypersensitivity. Association with thiazides and prior renal compromise. Arch Intern Med, 1974. 134 (3): p. 553-8. 64. Hayashi, T., et al., Inhibition of cow's milk xant hine oxidase by flavonoids. J Nat Prod, 1988. 51 (2): p. 345-8. 65. Nagao, A., M. Seki, and H. Kobayashi, Inhibition of xanthine oxidase by flavonoids. Biosci Biotechnol Biochem, 1999. 63 (10): p. 1787-90. 66. Zhu, J.X., et al., Effects of Biota orie ntalis extract and its flavonoid constituents, quercetin and rutin on serum uric acid l evels in oxonate-induced mice and xanthine dehydrogenase and xanthine oxid ase activities in mouse liver. J Ethnopharmacol, 2004. 93 (1): p. 133-40. 67. Donald voet, J.G.V., Biochemistry 3 ed. 2004: Von Hoffmann Corporation. 1096. 68. Haliwell, B., "Uric acid:an example of antio xidant evaluation"In handbook of antioxidants ed. E.P. Cadenas, L. 1996, Newyork: Marcel Dekker,Inc.

PAGE 146

146 69. Gennaro, A.R.C., G.D.;Marderosi an,A.;Harvey,S.C.;Hussar,D.A., Remington's Pharmaceutical Sciences 18 ed. 1990: Mack Publishing Company, USA. 70. Schlesinger, N., Management of acute and chronic gouty arthritis: presen t state-of-theart. Drugs, 2004. 64 (21): p. 2399-416. 71. Rott, K.T. and C.A. Agudelo, Gout. Jama, 2003. 289 (21): p. 2857-60. 72. Kim, K.Y., et al., A literature review of the epidemio logy and treatment of acute gout. Clin Ther, 2003. 25 (6): p. 1593-617. 73. Arromdee, E., et al., Epidemiology of gout: is the incidence rising? J Rheumatol, 2002. 29 (11): p. 2403-6. 74. Star, V.L. and M.C. Hochberg, Prevention and management of gout. Drugs, 1993. 45 (2): p. 212-22. 75. Donald voet, J.G.V., Biochemistry 3 ed. 2004: Von Hoffmann Corporation. 477-486. 76. Jang, G.R., R.Z. Harris, and D.T. Lau, Pharmacokinetics and its role in small molecule drug discovery research. Med Res Rev, 2001. 21 (5): p. 382-96. 77. Derendorf, H., et al., Pharmacokinetic/pharmacodynamic modeling in drug research and development. J Clin Pharmacol, 2000. 40 (12 Pt 2): p. 1399-418. 78. Leon Shargel, S.W.-P., Andrew B.C. Yu, Applied biopharmaceutics and pharmacokinetics 5 ed. 2005: The McGraw-Hill Companies, Inc. 79. Emmerson, B.T., The management of gout. Drug Therapy, 1996. 334 (7): p. 445-451. 80. JOel G. Hardman, A.G.G., Lee E.Limbird, Goodman & Gilman's The Pharmacological Basis of Therapeutics 9 ed. 1995: R.R Donnelley and Sons Company. 81. Walgren, R.A., et al., Cellular uptake of dietary flavonoi d quercetin 4'-beta-glucoside by sodium-dependent glucose transporter SGLT1. J Pharmacol Exp Ther, 2000. 294 (3): p. 837-43. 82. Schutz, K., et al., Quantitative determination of pheno lic compounds in artichoke-based dietary supplements and pharmaceuticals by high-performance liquid chromatography. J Agric Food Chem, 2006. 54 (23): p. 8812-7. 83. Fritsch, P.O. and A. Sidoroff, Drug-induced Stevens-Johnson syndrome/toxic epidermal necrolysis. Am J Clin Dermatol, 2000. 1 (6): p. 349-60. 84. Horiuchi, H., et al., Allopurinol induces renal toxic ity by impairing pyrimidine metabolism in mice. Life Sci, 2000. 66 (21): p. 2051-70. 85. Pereira, S., et al., [Fatal liver necrosis due to allopurinol]. Acta Med Port, 1998. 11 (12): p. 1141-4.

PAGE 147

147 86. Chan, W.S., P.C. Wen, and H.C. Chiang, Structure-activity relati onship of caffeic acid analogues on xanthine oxidase inhibition. Anticancer Res, 1995. 15 (3): p. 703-7. 87. Van Hoorn, D.E., et al., Accurate prediction of xanthine oxidase inhibition based on the structure of flavonoids. Eur J Pharmacol, 2002. 451 (2): p. 111-8. 88. Chiang, H.C., Y.J. Lo, and F.J. Lu, Xanthine oxidase inhibitors from the leaves of Alsophila spinulosa (Hook) Tryon. J Enzyme Inhib, 1994. 8 (1): p. 61-71. 89. Nguyen, M.T., et al., Xanthine oxidase inhibitors from the flowers of Chrysanthemum sinense. Planta Med, 2006. 72 (1): p. 46-51. 90. Cos, P., et al., Structure-activity relations hip and classification of flavonoids as inhibitors of xanthine oxidase and superoxide scavengers. J Nat Prod, 1998. 61 (1): p. 71-6. 91. Cimanga, K., Constituents from Morinda morindoides le aves as inhibitors of xanthine oxidase and scavengers of superoxide anions. Pharm. Pharmacol. Commum., 1999. 5 : p. 419-424. 92. Lin, C.M., et al., Molecular modeling of flavonoids that inhib its xanthine oxidase. Biochem Biophys Res Commun, 2002. 294 (1): p. 167-72. 93. Chang, W.S., et al., Inhibitory effects of flav onoids on xanthine oxidase. Anticancer Res, 1993. 13 (6A): p. 2165-70. 94. Noro, T., et al., Inhibitors of xanthine oxidase from the flowers and buds of Daphne genkwa. Chem Pharm Bull (Tokyo), 1983. 31 (11): p. 3984-7. 95. Chen, X., et al., Pharmacokinetics and modeling of quercetin and metabolites. Pharm Res, 2005. 22 (6): p. 892-901. 96. Boersma, M.G., et al., Regioselectivity of phase II met abolism of luteolin and quercetin by UDP-glucuronosyl transferases. Chem Res Toxicol, 2002. 15 (5): p. 662-70. 97. Hall, I.H., et al., Substituted cyclic imides as potential anti-gout agents. Life Sci, 1990. 46 (26): p. 1923-7. 98. Stavric, B., et al., Some in vivo effects in the ra t induced by chlorprothixene and potassium oxonate. Pharmacol Res Commun, 1975. 7 (2): p. 117-24. 99. Iwamoto, T., M. Yoshiura, and K. Iriyama, A simple, rapid and sensitive method for the determination of rat serum uric acid by reversed-phase high-performance liquid chromatography with elect rochemical detection. J Chromatogr, 1983. 278 (1): p. 156-9. 100. Masahiko Yoshiura TI, K.I., Toshinori Ka nki, Yasuo Kato, Hiroaki Sekino, and Norio Nakamura, A simple and sensitive method for the determination of uric acid in human cerebrospinal fluid by reve rsed-phase high-performance liquid chromatography with electrochemical detection. Jikeikai MedJ, 1983. 30 : p. 235-238.

PAGE 148

148 101. Johnson, W.J., B. Stavric, and A. Chartrand, Uricase inhibition in the rat by s-triazines: an animal model for hyperuricemia and hyperuricosuria. Proc Soc Exp Biol Med, 1969. 131 (1): p. 8-12. 102. Osada, Y., et al., Hypouricemic effect of the novel xanthine oxidase inhibitor, TEI-6720, in rodents. Eur J Pharmacol, 1993. 241 (2-3): p. 183-8. 103. Hollman, P.C., et al., Absorption of dietary quercetin gl ycosides and quercetin in healthy ileostomy volunteers. Am J Clin Nutr, 1995. 62 (6): p. 1276-82. 104. Shimoi, K., et al., Intestinal absorption of luteolin and lu teolin 7-O-beta-glucoside in rats and humans. FEBS Lett, 1998. 438 (3): p. 220-4. 105. Romanova, D., et al., Determination of apigenin in ra t plasma by high-performance liquid chromatography. J Chromatogr A, 2000. 870 (1-2): p. 463-7. 106. Di Carlo, G., et al., Inhibition of intestinal motility and secretion by flavonoids in mice and rats: structure-activity relationships. J Pharm Pharmacol, 1993. 45 (12): p. 1054-9. 107. Yu, Z., W.P. Fong, and C.H. Cheng, The dual actions of morin (3,5,7,2',4'pentahydroxyflavone) as a hypouricemic agent : uricosuric effect and xanthine oxidase inhibitory activity. J Pharmacol Exp Ther, 2006. 316 (1): p. 169-75. 108. Halliwell, B., Gutteridge, J.M.C, Free radicals in biology and medicine 2nd ed. 1989: Clarendron Press, Oxford. 422-437. 109. Holers, C.J.S.V.M., Gout, Hyperuricemia, and other Cr ystal-Associated Arthropathies 1999: Marcel Dekker, Inc. 1-4. 110. Dalbeth, N. and D.O. Haskard, Mechanisms of inflammation in gout. Rheumatology (Oxford), 2005. 44 (9): p. 1090-6. 111. Ou, B., M. Hampsch-Woodill, and R.L. Prior, Development and validation of an improved oxygen radical absorbance capacity as say using fluorescein as the fluorescent probe. J Agric Food Chem, 2001. 49 (10): p. 4619-26. 112. Manach, C., et al., Bioavailability and bioe fficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr, 2005. 81 (1 Suppl): p. 230S-242S. 113. Moon, J.H., et al., Accumulation of quercetin conjugates in blood plasma after the shortterm ingestion of onion by women. Am J Physiol Regul Integr Comp Physiol, 2000. 279 (2): p. R461-7. 114. Kong, A.N., et al., Induction of xenobiotic enzymes by the MAP kinase pathway and the antioxidant or electrophile response element (ARE/EpRE). Drug Metab Rev, 2001. 33 (34): p. 255-71.

PAGE 149

149 115. Walle, U.K. and T. Walle, Induction of human UDP-glucu ronosyltransferase UGT1A1 by flavonoids-struct ural requirements. Drug Metab Dispos, 2002. 30 (5): p. 564-9. 116. Natsume, M., et al., In vitro antioxidative activity of (-)-epicatechin glucuronide metabolites present in human and rat plasma. Free Radic Res, 2004. 38 (12): p. 1341-8. 117. Justino, G.C., et al., Plasma quercetin metabolites: structure-antioxidant activity relationships. Arch Biochem Biophys, 2004. 432 (1): p. 109-21. 118. King, R.A. and D.B. Bursill, Plasma and urinary kinetics of the isoflavones daidzein and genistein after a single soy meal in humans. Am J Clin Nutr, 1998. 67 (5): p. 867-72. 119. Ward, N.C., et al., Supplementation with grape seed polyphenols results in increased urinary excretion of 3-hydroxyphenylpr opionic Acid, an important metabolite of proanthocyanidins in humans. J Agric Food Chem, 2004. 52 (17): p. 5545-9. 120. Rios, L.Y., et al., Chocolate intake increases urinary excretion of polyphenol-derived phenolic acids in healthy human subjects. Am J Clin Nutr, 2003. 77 (4): p. 912-8. 121. Nagao, M., et al., Mutagenicities of 61 flavonoids and 11 related compounds. Environ Mutagen, 1981. 3 (4): p. 401-19. 122. Yasukawa, K., et al., Effect of chemical constituents from plants on 12-Otetradecanoylphorbol-13acetate-induced inflammation in mice. Chem Pharm Bull (Tokyo), 1989. 37 (4): p. 1071-3. 123. Ferriola, P.C., V. Cody, and E. Middleton, Jr., Protein kinase C inhibition by plant flavonoids. Kinetic mechanisms and structure-activity relationships. Biochem Pharmacol, 1989. 38 (10): p. 1617-24. 124. Wittemer, S.M. and M. Veit, Validated method for the determ ination of six metabolites derived from artichoke leaf extract in human plasma by high-performance liquid chromatography-coulometric-array detection. J Chromatogr B Anal yt Technol Biomed Life Sci, 2003. 793 (2): p. 367-75. 125. Li, L., et al., Simultaneous determination of luteo lin and apigenin in dog plasma by RPHPLC. J Pharm Biomed Anal, 2005. 37 (3): p. 615-20. 126. Cova D, D.A.L., Giavarini F, Palladini G, and Perego R, Pharmacokinetics and metabolism of oral diosmin in healthy volunteers. Int J Clin Pharmacol Ther Toxicol, 1992. 30 : p. 29-33. 127. Boutin, J.A., et al., In vivo and in vitro glucuronidation of the flavonoid diosmetin in rats. Drug Metab Dispos, 1993. 21 (6): p. 1157-66. 128. Ader, P., A. Wessmann, and S. Wolffram, Bioavailability and metabolism of the flavonol quercetin in the pig. Free Radic Biol Med, 2000. 28 (7): p. 1056-67.

PAGE 150

150 129. Murota, K., et al., Unique uptake and transport of isoflavone aglycones by human intestinal caco-2 ce lls: comparison of isof lavonoids and flavonoids. J Nutr, 2002. 132 (7): p. 1956-61. 130. Nielsen, S.E., et al., Effect of parsley (Petroselinum crispum) intake on urinary apigenin excretion, blood antioxidant enzymes and bi omarkers for oxidative stress in human subjects. Br J Nutr, 1999. 81 (6): p. 447-55. 131. Gugler, R., M. Leschik, and H.J. Dengler, Disposition of quercetin in man after single oral and intravenous doses. Eur J Clin Pharmacol, 1975. 9 (2-3): p. 229-34. 132. Ferry, D.R., et al., Phase I clinical trial of the fla vonoid quercetin: pharmacokinetics and evidence for in vivo ty rosine kinase inhibition. Clin Cancer Res, 1996. 2 (4): p. 659-68. 133. Ma, Y., et al., LC/MS/MS quantitation assay for pharmacokinetics of naringenin and double peaks phenomenon in rats plasma. Int J Pharm, 2006. 307 (2): p. 292-9.

PAGE 151

151 BIOGRAPHICAL SKETCH Sasiporn Sarawek was born in April 14th, 1978, in Chiangmai, Thailand. She obtained her bachelors degree in Pharmacy in 2001 from Chiangmai University. She started her PhD program in January 2003 in the Department of Pharmaceutics of the University of Florida under supervision of Dr. Veronika Butterweck and Dr Hartmut Derendorf. Sasiporn received her PhD in Pharmaceutics in August 2007.