PHYTOCHEMICAL AND BIOLOGICAL INVESTIGATION ON PAEONIA EMODI WALL. EX ROYL AND BERGENIA LIGULATA SENSU BLATTER Ph.D. Thesis By ANWAR SADAT INSTITUTE OF CHEMICAL SCIENCES UNIVERSITY OF PESHAWAR, PESHAWARPAKISTAN, JANUARY 2014 PHYTOCHEMICAL AND BIOLOGICAL INVESTIGATION ON PAEONIA EMODI WALL. EX ROYL AND BERGENIA LIGULATA SENSU BLATTER By Anwar Sadat Dissertation submitted to the University of Peshawar in partial fulfillment of the requirements for the degree of doctor of philosophy in organic chemistry INSTITUTE OF CHEMICAL SCIENCES UNIVERSITY OF PESHAWAR, PESHAWARPAKISTAN, JANUARY 2014 Dedicated to my parents, my family & teachers TABLE OF CONTENTS Acknowledgments---------------------------------------------------------------------------- I Abstract---------------------------------------------------------------------------------------- iii List of Abbreviations------------------------------------------------------------------------- v List of Schemes------------------------------------------------------------------------------- vi List of Tables---------------------------------------------------------------------------------- vii List of Figures--------------------------------------------------------------------------------- ix Chapter 1 General introduction------------------------------------------------------------------------ 1 Chapter 2 Biosynthesis----------------------------------------------------------------------------------- 5 2.1. Primary metabolites--------------------------------------------------------------------- 5 2.2. Secondary metabolites------------------------------------------------------------------ 6 2.3. Biosynthesis of flavonoids------------------------------------------------------------- 8 Chapter 3 Plant Introduction--------------------------------------------------------------------------- 15 3.1 Plant Introduction------------------------------------------------------------------------ 15 3.1.1. Family Paeoniaceae--------------------------------------------------------------- 16 3.1.2. Genus Paeonia-------------------------------------------------------------------- 16 3.1.3. Paeonia emodi Wall. Ex Royle------------------------------------------------- 16 3.2. Pharmacological importance of the Genus Paeonia-------------------------------- 16 3.3. Literature survey on the genus Paeonia---------------------------------------------- 19 3.4. Structures of selected compound reported from genus Paeonia------------------ 32 Chapter 4 Results & Discussion------------------------------------------------------------------------ 40 4.1. New constituents isolated from P. emodi------------------------------------------- 40 4.2. Hitherto unreported constituents from P. emodi---------------------------------- 40 4.3. Known constituents from P. emodi------------------------------------------------- 41 4.4. Structure elucidation of new compounds from P. emodi----------------------- 42 4.4.1. Paenoiflorin C (6)---------------------------------------------------------------- 42 4.4.2. Paeoniflorin D (7)---------------------------------------------------------------- 46 4.4.3. Kaempferol-3-O-β-D-glucopyranosyl-7-oic acid (12)---------------------- 50 4.4.4. Kaempferol-7-al-3-O-β-D-glucopyranoside (15)---------------------------- 54 4.4.5. Kaempferol-7-methoxy-3-O-β-D-glucopyranosyl-3'-oic acid (16)------- 58 4.5. Hitherto unreported compounds----------------------------------------------------- 62 4.5.1. 2,3-Dihydroxy-4-methoxyacetophenone (3) 62 4.5.2 Kaempferol-3’-methoxy-3-β-D-glucopyranoside (5)----------------------- 64 4.5.3. Debenzoyl-8-galloylpaeoniflorin (8)------------------------------------------ 67 4.5.4. 2-O-Ethyl-β-D-glucopyranoside (9)------------------------------------------- 70 4.5.5. 2,3-Dihydro-5-hydroxy-6-methyl-3-benzofuranmethanol (17)---------- 72 4.5.6. p-Digallic acid (18)--------------------------------------------------------------- 74 4.6. Known compounds from P. emodi---------------------------------------------- 76 4.6.1. Ethyl gallate (1)------------------------------------------------------------------ 76 4.6.2. Methyl gallate (2)---------------------------------------------------------------- 78 4.6.3.Debenzoylpaeoniflorin-4-methylether-8-benzoyl-1-O-β-D-gluco- pyranoside (4)---------------------------------------------------------------------------- 80 4.6.4. Quercetin-3-β-D-glucopyranoside (10)--------------------------------------- 83 4.6.5. Kaempferol-3-β-D-glucopyranoside (Astragalin) (11)--------------------- 86 4.6.6. 8-Debenzoylpaeoniflorin-1-O-β-D-glucopyranoside (13)----------------- 89 4.6.7. Debenzoyl-8-galloylpaeoniflorin-1-O-β-D-glucopyranoside (14)-------- 92 4.7. DPPH antioxidant assay---------------------------------------------------------------- 96 4.8. Antibacterial activities ----------------------------------------------------------------- 97 4.9. Antifungal activities--------------------------------------------------------------------- 97 4.10. Urease inhibition assay---------------------------------------------------------------- 98 4.11. Brine shrimp (Artemia salina) lethality bioassay--------------------------------- 100 4.12. Antiplasmodial activity--------------------------------------------------------------- 101 Chapter 5 Experimental (Part A)---------------------------------------------------------------------- 102 5.1. General experimental condition------------------------------------------------------ 102 5.1.1. Physical contents----------------------------------------------------------------- 102 5.1.2. Chromatographic techniques--------------------------------------------------- 103 5.2. Materials and methods----------------------------------------------------------------- 103 5.2.1. Plant material--------------------------------------------------------------------- 103 5.2.2. Extraction and isolation-------------------------------------------------------- 104 5.2.3. Acid hydrolysis------------------------------------------------------------------- 106 5.3. Characterization of new compounds from P. emodi------------------------------ 109 5.3.1. Paeoniflorin C (6)---------------------------------------------------------------- 109 5.3.2. Paeoniflorin D (7)---------------------------------------------------------------- 109 5.3.3. Kaempferol-3-O-β-D-glucopyranosyl-7-oic acid (12)---------------------- 110 5.3.4. Kaempferol-7-al-3-O-β-D-glucopyranosid (15)----------------------------- 110 5.3.5. Kaempferol-7-methoxy-3-O-β-D-glucopyranosyl-3ʹ-oic acid (16)------- 111 5.4. Hitherto unreported constituents from P. emodi---------------------------------- 112 5.4.1. 2,3-Dihydroxy-4-methoxyacetophenone (3)-------------------------------- 112 5.4.2. Quercetin-3'-methoxy-3-β-D-glucopyranoside (5)------------------------- 112 5.4.3. Debenzoyl-8-galloylpaeoniflorin (8)------------------------------------------ 113 5.4.4. 2-O-Ethyl-β-D-glucopyranoside (9)------------------------------------------- 113 5.4.5. 2,3-Dihydro-5-hydroxy-6-methyl-3-benzofuranmethanol (17)------------- 114 5.4.6. p-Digallic acid (18)--------------------------------------------------------------- 114 5.5. Known constituents from P. emodi--------------------------------------------------- 115 5.5.1. Ethyl gallate (1)------------------------------------------------------------------- 115 5.5.2. Methyl gallate (2)----------------------------------------------------------------- 115 5.5.3. Debenzoylpaeoniflorin; 4-methylether-8-benzoyl-1-O- β-D-glucopyranoside (4)-------------------------------------------------------- 116 5.5.4. Quercetin-3-β-D-glucopyranoside (10)--------------------------------------- 116 5.5.5. Kaempferol-3-β-D-glucopyranoside (astragalin) (11)---------------------- 117 5.5.6. 8-Debenzoylpaeoniflorin-1-O-β-D-glucopyranoside (13)----------------- 117 5.5.7. Debenzoyl-8-galloylpaeoniflorin-1-O-β-D-glucopyranoside (14)-------- 118 5.6. Biological screening-------------------------------------------------------------------- 119 5.6.1. DPPH antioxidant assay-------------------------------------------------------- 119 5.6.2. Antibacterial activities---------------------------------------------------------- 119 5.6.3. Antifungal activities---------------------------------------------------------- 119 5.6.3. Brine shrimp (Artemia salina) lethality bioassay--------------------------- 121 5.6.4. Urease inhibition assay--------------------------------------------------------- 121 5.6.5. Antiplasmodial activity--------------------------------------------------------- 122 Chapter 6 Introduction (Part B)----------------------------------------------------------------------- 124 6.1. Plant introduction------------------------------------------------------------------------ 124 6.1.1. Saxifragaceae---------------------------------------------------------------------- 125 6.1.2. Genus Bergenia------------------------------------------------------------------- 125 6.1.3. Bergenia ligulata------------------------------------------------------------------ 125 6.2. Pharmacological importance of the genus Bergenia------------------------------- 126 6.3. Literature survey of the genus Bergenia---------------------------------------------- 127 6.4. Structure of selected compounds reported from genus Bergenia----------------- 129 Chapter 7 Results & Discussion------------------------------------------------------------------------ 137 7.1. New constituents from B. Ligulata---------------------------------------------------- 137 7.2. Hitherto unreported constituents from B. Ligulata----------------------------------