PHARMACOKINETIC AND PHARMACODYNAMIC pharmacokinetic parameters were observed for each stereoisomeric

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  • PHARMACOKINETIC AND PHARMACODYNAMIC INVESTIGATIONS OF

    SELECTED CHIRAL FLAVONOIDS

    By

    Karina R. Vega-Villa

    A dissertation submitted in partial fulfillment of the requirements for the degree of

    DOCTOR OF PHILOSOPHY

    WASHINGTON STATE UNIVERSITY

    College of Pharmacy

    May 2009

  • iii

    ACKNOWLEDGEMENTS

    There have been many people who contributed to the research presented in this thesis.

    First and foremost, I would like to express my deepest gratitude to my thesis advisor, Dr. Neal

    M. Davies, for his exceptional guidance and scientific expertise during my time at

    Washington State University. Thanks for your support and encouragement, both scientifically

    and personally.

    Special thanks to my Ph.D. Committee: Dr. Catherine Elstad, Dr. Arash Hatefi, Dr.

    Steve Martinez, and Dr. Preston Andrews for their support and guidance in the preparation of

    this thesis. I would also like to acknowledge Dr. Lisa Gloss and Traci Topping for assistance

    and use of their circular dichroism spectrometer.

    I wish to thank all my colleagues in the laboratory: Connie M. Remsberg, Jody K.

    Takemoto, Nicole, D. Miranda and Dr. Yusuke Ohgami. Thanks for creating a great

    environment at work and for your friendship and moral support. Special thanks to past lab

    members, and especially to Dr. Jaime A. Yáñez F. for his time and patience in teaching me

    analytical and cell culture techniques. I would also like to acknowledge Marilyn Sánchez-

    Bonilla, whose support and friendship helped me through graduate life in Pullman.

    Thanks to the staff and faculty in the Department of Pharmaceutical Sciences for

    always having their doors open and for helping me to find the answers I needed.

    I acknowledge the support of my graduate research awards: the Dorothy Otto Kennedy

    Scholarship Fund, the Travel Award for Graduate Students from the Western Pharmacology

    Society, the Graduate School Travel Grant from Washington State University, the Ruben

    Loera Engineering and Science Award, and teaching assistantships from the Department of

    Pharmaceutical Sciences at Washington State University.

  • iv

    APPLICATIONS OF FLAVONOIDS ANALYSIS IN PHARMACEUTICAL AND

    HORTICULTURAL STUDIES

    ABSTRACT

    By Karina R. Vega-Villa, Ph.D.

    Washington State University

    May 2009

    Chair: Dr. Neal M. Davies

    High-performance liquid chromatographic (HPLC) methods were validated for the

    determination of homoeriodictyol, isosakuranetin, and taxifolin enantiomers in biological

    matrices. The quantification and racemization of these flavonoids in lemon, Yerba Santa,

    grapefruit, tu-fu-ling, tomato, and apple was accomplished.

    The validated HPLC methods were applied in studying the pharmacokinetics of

    homoeriodictyol, isosakuranetin, and taxifolin in a rodent animal model. Their stereoisomers

    were detected in serum and urine of rats primarily as glucuro-conjugates. Different

    pharmacokinetic parameters were observed for each stereoisomeric form for all three

    xenobiotics including: half-life, total clearance, volume of distribution, and area under the

    curve.

    To assess cytotoxicity, a variety of cancer cell lines were treated with racemic and

    stereoisomeric homoeriodictyol, isoskuranetin, and taxifolin. (+/-)-Isosakuranetin was most

    effective in cell growth inhibition of all cell lines studied. To assess amelioration of

    inflammation in an in vitro colitis model, colon adenocarcinoma cells were treated with the

    three flavonoids. Inflammation was induced and prostaglandin E2 (PGE2) release was

    measured. The flavonoids exhibited a concentration-dependent reduction in PGE2 levels. The

    three flavonoids were also assessed for their antioxidant capacity; and cyclooxygenases and

  • v

    histone deacetylase inhibitory activities. To evaluate experimental reduction of adipogenesis,

    pre-adipocytes were treated with the flavonoids. Differentiation was induced and triglyceride

    accumulation was assessed. The flavonoids showed concentration-dependent inhibition of

    triglyceride accumulation.

    Using an alternative HPLC method of analysis, separation and quantification of

    various polyphenols in tomatoes and apples was achieved. Concentrations of polyphenols

    were measured with higher concentrations found in peel. The pharmacological activity of

    apple extracts was subsequently examined. Peel extracts demonstrated cell growth inhibition;

    reduction of inflammatory markers in experimental arthritis and colitis models; anti-oxidant

    activity; and inhibition of triglyceride accumulation in adipocytes.

    In conclusion, the developed HPLC methods for the chiral flavonoids were sensitive,

    and stereospecific. Stereospecific assessment of the quantity of these flavonoids in selected

    fruit was also achieved. Stereoisomers of homoeriodictyol, isosakuranetin, and taxifolin were

    functionally distinct; enantio-specific pharmacokinetics and pharmacological activities were

    observed. Furthermore, analysis of polyphenol content was successfully achieved in tomatoes

    and apples; and the pharmacological activity of selected apple extracts demonstrated potential

    phytopreventive health benefits that require further experimental scrutinity.

  • vi

    TABLE OF CONTENTS

    ACKNOWLEDGEMENTS …………………………………………………………….….... iii

    ABSTRACT ………………………………………………………………………………......iv

    LIST OF TABLES ……………………………………………………………………...…...xix

    LIST OF FIGURES …………………………………………………………………….…..xxii

    PUBLICATIONS IN SUPPORT OF THIS THESIS ……………………………………....xxx

    ABBREVIATIONS AND SYMBOLS ………………………………………………….....xxxi

    CHAPTER 1

    1. LITERATURE REVIEW AND BACKGROUND…………………………………...…1

    1.1. INTRODUCTION …………………………………………………….………..…… 1

    1.2. BACKGROUND …………………..………………………………………...……… 2

    1.3. HOMOERIODICTYOL ……………..…………………………………..….………. 6

    1.3.1. Natural Sources ………………………………….…………………..….……. 6

    1.3.2. Commercial Uses……………………………….……………………….……. 6

    1.3.3. Biosynthesis …………………………………………………………...………7

    1.3.4. Current Methods of Analysis ………………………………………...………. 8

    1.3.5. Pharmacokinetic Studies …………………………………………..…….…… 9

    1.3.6. Pharmacological Activity …………………………………………..……….. 11

    1.4. ISOSAKURANETIN …………………………………………………….……...… 11

    1.4.1. Natural Sources …………………………………………………...……....… 11

    1.4.2. Commercial Uses ………………………………………………….…………12

  • vii

    1.4.3. Biosynthesis ……………………………………………………………....… 13

    1.4.4. Current Methods of Analysis …………………………………………......… 13

    1.4.5. Pharmacokinetic Studies ………………………………………………….… 14

    1.4.6. Pharmacological Activity ………………………………….………….......… 15

    1.5. TAXIFOLIN ……………………………………………………………….….....… 16

    1.5.1. Natural Sources ……………………………………………...……..…......… 16

    1.5.2. Commercial Uses ………………………………………………...….........… 18

    1.5.3. Biosynthesis ……………………………………………………...….........… 18

    1.5.4. Current Methods of Analysis …………………………………...…...……… 20

    1.5.5. Pharmacokinetic Studies ………………………………………...………….. 22

    1.5.6. Pharmacological Activity ………………………………………...…………. 24

    1.6. OBJECTIVES ………………………………………………..………….....…….… 25

    CHAPTER 2

    2. STEREOSPECIFIC ASSAY DEVELOPMENT AND VALIDATION OF

    HOMOERIODICTYOL ………………………………………………….……........….27

    2.1. INTRODUCTION …………………………………………………….…….…...… 27

    2.2. BACKGROUND …………………………………………………….…….………. 28

    2.3. METHODS ………………………………………………………….………....……28

    2.3.1. HPLC Apparatus and Conditions …………………………………………….28

    2.3.2. Chemicals and Reagents ……………………………………………….……

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