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Pharmacological Characterization of Platycodin D and Cimicoxib in Terms of Their

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Pharmacological Characterization of Platycodin D and Cimicoxib in Terms of Their A Dissertation for the Degree of Doctor of Philosophy Pharmacological Characterization of Platycodin D and Cimicoxib in Terms of Their Dynamic and Kinetic Profiles Department of Veterinary Medicine Graduate School Chungnam National University By Tae-Won Kim Advisor Hyo-In Yun & Mario Giorgi August, 2014 Pharmacological Characterization of Platycodin D and Cimicoxib in Terms of Their Dynamic and Kinetic Profiles Advisor Hyo-In Yun & Mario Giorgi Submitted to the Graduate School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy April, 2014 Department of Veterinary Medicine Graduate School Chungnam National University By Tae-Won Kim To Approve the Submitted Dissertation for the Degree of Doctor of Philosophy By Tae-Won Kim Title: Pharmacological Characterization of Platycodin D and Cimicoxib in Terms of Their Dynamic and Kinetic Profiles June, 2014 Committee Chair _______________________ Committee _______________________ Committee _______________________ Committee _______________________ Committee _______________________ Graduate School Chungnam National University CONTENTS Part I General introduction --------------------------------------------------------------- 1 Chapter 1. Anti-hepatitis C viral activity and hepatoprotective effect of standardized Platycodon grandiflorum extract on chemical-induced hepatotoxicity in mice Abstract ------------------------------------------------------------------------------ 5 Introduction ------------------------------------------------------------------------- 6 Materials and methods ----------------------------------------------------------- 9 Chemicals and reagents ------------------------------------------------------------ 9 Preparation of PG ------------------------------------------------------------------ 9 Gastric degradation simulation -------------------------------------------------- 10 HPLC-ELSD analysis ------------------------------------------------------------ 11 Anti-HCV activity in the HCV replicon model of PG ----------------------- 11 Cytotoxicity of PG ---------------------------------------------------------------- 13 Polyphenolic content and DPPH radical scavenging activity -------------- 13 Animals and treatments ---------------------------------------------------------- 14 Serum and hepatic biochemical examination --------------------------------- 15 Histopathological examination -------------------------------------------------- 16 Statistical analysis ---------------------------------------------------------------- 16 Results ------------------------------------------------------------------------------- 17 HPLC-ELSD fingerprint-analysis ---------------------------------------------- 17 Total phenol content and DPPH radical scavenging activity --------------- 18 Anti-HCV activity of PG ------------------------------------------------------- 20 i Effect of PG on serum liver enzymes ----------------------------------------- 21 Hepatic levels of GSH, TBARS, NO and SOD ------------------------------ 24 Histopathological observations ------------------------------------------------- 27 Discussion --------------------------------------------------------------------------- 31 References -------------------------------------------------------------------------- 37 Chapter 2. Protective effect of the standardized aqueous extract from the root of Platycodon grandiflorum on bile duct ligation-induced hepatic injury Abstract ----------------------------------------------------------------------------- 43 Introduction ------------------------------------------------------------------------ 45 Materials and methods ----------------------------------------------------------- 48 Chemicals and reagents ---------------------------------------------------------- 48 Animals and treatments ---------------------------------------------------------- 49 Serum biochemical examination ----------------------------------------------- 51 Oxidant and antioxidant parameters ------------------------------------------- 51 Measurement of plasma TGF-β1 levels --------------------------------------- 51 Histopathological examination -------------------------------------------------- 52 Statistical analysis ---------------------------------------------------------------- 52 Results ------------------------------------------------------------------------------- 53 Assessment of serum hepatic enzyme activities ----------------------------- 53 Effect of PG on GSH, SOD, TBARS, NO values --------------------------- 56 Histopathological findings ------------------------------------------------------- 59 Serum TGF- β level -------------------------------------------------------------- 63 Discussion -------------------------------------------------------------------------- 64 References -------------------------------------------------------------------------- 69 ii Chapter 3. Platycodin D attenuates bile duct ligation-induced hepatic injury and fibrosis in mice Abstract ---------------------------------------------------------------------------- 75 Introduction ------------------------------------------------------------------------ 76 Materials and methods ----------------------------------------------------------- 77 Chemicals and reagents --------------------------------------------------------- 77 Animals and treatments --------------------------------------------------------- 78 Serum biochemical examination ------------------------------------------------ 79 Determination of hepatic GSH, SOD, TBARS, and NO levels ----------- 80 Histopathological examination -------------------------------------------------- 80 Immunohistochemical analysis ------------------------------------------------- 81 Statistical analysis ---------------------------------------------------------------- 82 Results ------------------------------------------------------------------------------- 82 Serum hepatic enzyme activity ------------------------------------------------- 82 Evaluation of hepatic GSH, SOD, TBARS, and NO levels ----------------- 84 Histopathological findings ------------------------------------------------------- 86 Immunohistochemical analysis ------------------------------------------------- 89 Discussion --------------------------------------------------------------------------- 92 References -------------------------------------------------------------------------- 97 Chapter 4. Platycodin D-induced apoptosis in CT26 cells and its synergistic effect with cisplatin on tumor bearing mice Abstract --------------------------------------------------------------------------- 101 Introduction ---------------------------------------------------------------------- 102 Materials and methods --------------------------------------------------------- 103 Chemicals and reagents --------------------------------------------------------- 103 In vitro study --------------------------------------------------------------------- 104 iii Determination of combination index --------------------------------------- 104 Cell cycle analysis ------------------------------------------------------------- 105 Apoptosis assay ---------------------------------------------------------------- 106 Western blot analysis --------------------------------------------------------- 107 In vivo study --------------------------------------------------------------------- 107 Animals and treatments ------------------------------------------------------- 107 Determination of synergistic index ----------------------------------------- 109 Serum biochemical examination --------------------------------------------- 109 Oxidative status in liver and kidney tissue --------------------------------- 110 Pharmacokinetic study --------------------------------------------------------- 110 Statistical analysis --------------------------------------------------------------- 112 Results ----------------------------------------------------------------------------- 112 In vitro study --------------------------------------------------------------------- 112 Synergism assay ---------------------------------------------------------------- 112 Cell cycle and apoptosis analysis ------------------------------------------- 115 Western blot analysis --------------------------------------------------------- 118 In vivo study ---------------------------------------------------------------------- 119 Determination of synergistic index ----------------------------------------- 119 Serum biochemical examination --------------------------------------------- 121 Evaluation of GSH, SOD, TBARS, and NO levels ---------------------- 123 Pharmacokinetic analysis ------------------------------------------------------ 125 Discussion ------------------------------------------------------------------------- 127 References ------------------------------------------------------------------------- 132 General conclusion -------------------------------------------------------------- 138 iv Part II General Introduction ---------------------------------------------------------- 140 Chapter 1. Pharmacokinetic profiles of the novel COX-2 selective inhibitor cimicoxib in dogs Abstract --------------------------------------------------------------------------- 165 Introduction ---------------------------------------------------------------------- 166 Materials and methods --------------------------------------------------------- 168 Chemical and reagents --------------------------------------------------------- 168 Animal studies -------------------------------------------------------------------
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