STUDIES ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF CONOCARPUS LANCIFOLIUS (COMBRETACEAE) By MALIK SAADULLAH 01-PhDC-12 Session 2012-2015 A thesis submitted in Partial fulfillment of the requirements for the degree of Doctorate of Philosophy in Pharmaceutical Chemistry FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY, MULTAN CERTIFICATE This is to certify that the thesis entitled, “STUDIES ON CHEMICAL CONSTITUENTS AND BIOLOGICAL ACTIVITIES OF CONOCARPUS LANCIFOLIUS (COMBRETACEAE)” submitted by Mr. MALIK SAADULLAH has been thoroughly studied. It is found to be prolific in scope and quality as a thesis for the award of degree of Doctor of Philosophy in Pharmacy (Pharmaceutical Chemistry). (Prof. Dr. Bashir Ahmad Ch.) Supervisor Dedicated to my Beloved Ones ACKNOWLEDGEMENT I have no words at my command to express my deepest sense of gratitude to Almighty Allah, the compassionate, the merciful, who enabled me to complete my research work. My special praises are for Holy Prophet, Hazarat Muhammad (Peace Be Upon Him) who is, forever, a source of guidance and knowledge for the whole humanity. It is a matter of great pleasure and honour for me to express my heartiest gratitude and appreciation to my respectable and honourable supervisor Dr. Bashir Ahmad Ch. Professor of Pharmaceutical chemistry Faculty of Pharmacy, Bahauddin Zakariya University, Multan, for his valuable and ever inspiring guidance, persistent encouragement and sagacious advice provided to me throughout this research work. With a deep sense of gratitude I communicate my heartful gratifications to Dr. Muhammad Uzair professor of pharmaceutical chemistry Faculty of Pharmacy, Bahauddin Zakariya University, Multan for his encouragement, useful suggestions and inspiring help during the conduct of my research work. I am grateful to Dr. Shazia Anjum Professor, Department of Chemistry, The Islamia University Bahawalpur for her kind guidance and help in my research work. I am indebted of heartiest thanks and deepest regards for lab staff for their help and immense cooperation for providing me chemicals and equipments. MALIK SAADULLAH BRIEF CONTENTS Serial No. Contents Page No. 1 List of Contents І 2 List of Abbreviations V 3 List of Figures vіі 4 List of Tables Іx 5 Aims of the present work 1 6 Abstract 2 7 Introduction 6 8 Literature Review 21 9 Materials and Methods 75 10 Results and Discussions 98 11 References 158 LIST OF CONTENTS S.No Desription Page AIM OF THE PRESENT WORK 1 ABSTRACT 2 1 Introduction 6 1.1 Different medicine systems 9 1.1.1 European system of medicines 9 1.1.2 Chinese system of medicine 9 1.1.3 Egyptian system of medicine 9 1.1.4 Ayurvedic System of Medicine 9 1.1.5 Unani System of Medicines 9 1.2 Herbal Medicines in Islam 10 1.3 Importance of Herbal Medicines 10 1.4 Herbal Medicines Today 10 1.5 Introduction to family combretaceae 13 1.6 Botanical aspects of genus Conocarpus 13 1.7 Conocarpus lancifolius 13 1.7.1 Botanical classification of Conocarpus lancifolius 14 1.8 Secondary metabolites 14 1.8.1 Alkaloids 16 1.8.2 Steroids 17 1.8.3 Terpenoids 18 1.8.4 Tannins 18 1.8.5 Glycosides 19 2 Literature review 21 2.1 Previous reports on the phytochemical studies from various species 21 of genera Terminalia, Combretum and Conocarpus. 2.2 Previous reports on the biological studies from various species of 53 genera Terminalia, Combretum and Conocarpus. i 3 Materials and Methods 75 3.1 Plant material 75 3.2 Solvents and chemicals 75 3.3 Preparation of reagents and solutions 75 3.4 Equipments/ instruments/ glasswares 77 3.5 Phytochemical methods 78 3.5.1 Extraction 78 3.5.2 Phytochemical screening 78 3.5.3 Techniques for chromatography 81 3.5.3.1 Thin layer chromatography 81 3.5.3.2 Column chromatography 83 3.6 Biological methods 83 3.6.1 Brine shrimp lethality assay 83 3.6.2 Antibacterial assay 84 3.6.3 Antifungal assay 85 3.6.4 Phytotoxic bioassay 85 3.6.5 Leishmanicidal assay 86 3.6.6 Antioxidant assay 86 3.6.7 Acetylcholinesterase assay 86 3.6.8 Butyrylcholinesterase assay 87 3.6.9 α- Chymotrypsin assay 87 3.6.10 Lipoxygenase assay 88 3.6.11 Urease assay 88 3.6.12 Alpha glucosidase assay 88 3.7 Spectroscopic methods 89 3.8 Physical and spectroscopic data of isolated compounds A-G 90 4 Results and discussions 98 4.1 Phytochemical studies of Conocarpus lancifolius 98 4.1.1 Extraction 98 4.1.2 Phytochemical screening for secondary metabolites 98 4.2 Isolation of compounds A-G 99 4.3 Structural elucidation of isolated compounds 102 4.4 Biological studies of Conocarpus lancifolius 140 ii 4.4.1 Acetylcholinesterase inhibition activities 140 4.4.2 Butyrylcholinesterase inhibition activities 141 4.4.3 Lipoxygenase inhibition activities 141 4.4.4 Carbonic anhydrase inhibition activities 142 4.4.5 Xanthine oxidase inhibition activities 142 4.4.6 β-glucuronidase inhibition activities 143 4.4.7 α-Chymotrypsin inhibition activities 144 4.4.8 Urease inhibition activities 144 4.4.9 Alpha glucosidase inhibitory activities 145 4.4.10 Blood glucose level 146 4.4.11 Hypolipidemic effect 146 4.4.12 Effect on liver function tests (SGPT & SGOT values) 148 4.4.13 In vitro screening on isolated tissue preparation 148 4.4.14 Antimicrobial activity 149 4.4.15 Antifungal activity 150 4.4.16 Phytotoxic assay 150 4.4.17 Brine shrimp lethality assay 151 4.4.18 Antileishmanial activity 152 4.4.19 Antioxidant activity 152 4.5 Total Flavonoid Contents 153 4.6 Quantification of phenolic contents by HPLC 153 4.7 Evaluation of biological activities of isolated compounds 154 5 References 158 iii LIST OF ABBRIVIATIONS µl Microlitre µm Micrometer 1, 2, 3, 4, 5, 6,------ Symbols for compounds reported from literature 12(S)-HETE 12-hydroxyeicosatetraenoic acid 12-HHTrE 12-hydroxyheptadecatrienoic acid 13C-NMR Carbon13 nuclear magnetic resonance spectroscopy 1H-NMR Proton nuclear magnetic resonance spectroscopy 5(S)-HETE 5-hydroxyeicosatetraenoic acid A, B, ----- Symbols for compounds isolated in this study AA Arachidonic acid ALP Alkaline phosphatase ALT Alanine transaminase AST Aspartate transaminase BHT Butylated hydroxy toluene BP British pharmacopoeia CCL4 Carbon tetrachloride DCM Dichloromethane DEPT Distortionless enhancement by polarization transfer DMSO Dimethyl sulfoxide DNFB Dinitrofluorobenzene DPPH Diphenyl picryl hydrazide E.coli Escherchia coli Edn Edition EIMS Electron impact mass spectrometry EtOH Ethanol HLE Human leukocyte elastase HMBC Heteronuclear multiple bond correlation HPLC High performance liquid chromatography HR- EIMS High resolution electron impact mass spectrometry HSQC Heteronuclear spin quantum correlation IC50 Inhibitory concentration50 iv LCT Long chain triglyceride LD50 Lethal dose50 LTB4 Leukotriene B4 MeOH Methanol MHZ Megahertz MIC Minimum inhibitory concentration. mmol/L Millimole per litre nm Nanometer NOE Nuclear overhauser effect PMNL Polymorphonuclear leukocytes PMSF Phenyl methyl sulfonyl fluoride SGOT Serum glutamate oxaloacetate transaminase SGPT Serum glutamate pyruvate transaminase TLC Thin layer chromatography TPA Tetradecanoylphorbol acetate USP United state pharmacopoeia UV Ultraviolet v LIST OF FIGURES S. No. Description Page Figure 1.1 Aerial view of the plant Conocarpus lancifolius 14 Figure 1.2 Interrelationships of biosynthetic pathways leading to 15 secondary constituents in plants. Figure 2.1 Chemical structures of flavonoids (1-51) reported from 23 various species of genera Terminalia, Combretum and Conocarpus. Figure 2.2 Chemical structures of terpens (52-119) reported from various 30 species of genera Terminalia, Combretum and Conocarpus. Figure 2.3 Chemical structures of tannins (120-138) reported from 40 various species of genera Terminalia, Combretum and Conocarpus Figure 2.4 Chemical structures of glycosides (139-164) reported from 46 various species of genera Terminalia, Combretum and Conocarpus. Figure 2.5 Chemical structures of mislinious compounds (165-178) 51 reported from various species of genera Terminalia, Combretum and Conocarpus Figure 4.1 Isolation of compounds A-D from the dichloromethane 100 extract of Conocarpus lancifolius. Figure 4.2 Isolation of compounds E-G from the methanol extract of 101 Conocarpus lancifolius. Figure 4.3 UV spectrum of compound A 103 Figure 4.4 IR spectrum of compound A 103 Figure 4.5 1H-NMR spectrum of compound A 104 Figure 4.6 13C-NMR (BB) spectrum of compound A 104 Figure 4.7 13C NMR (dept) spectrum of compound A 105 Figure 4.8 EI spectrum of compound A 105 Figure 4.9 FAB+ spectrum of compound A 106 Figure 4.10 FAB- spectrum of compound A 106 Figure 4.11 UV spectrum of compound B 108 vi Figure 4.12 IR spectrum of compound B 108 Figure 4.13 1H-NMR spectrum of compound B 109 Figure 4.14 13C-NMR (BB) spectrum of compound B 109 Figure 4.15 13C NMR (dept) spectrum of compound B 110 Figure 4.16 EI spectrum of compound B 110 Figure 4.17 FAB+ spectrum of compound B 111 Figure 4.18 COSY spectrum of compound B 111 Figure 4.19 HSQC spectrum of compound B 112 Figure 4.20 UV spectrum of compound C 114 Figure 4.21 IR spectrum of compound C 114 Figure 4.22 1H-NMR spectrum of compound C 115 Figure 4.23 13C-NMR (BB) spectrum of compound C 115 Figure 4.24 13C NMR (dept) spectrum of compound C 116 Figure 4.25 EI spectrum of compound C 116 Figure 4.26 FAB+ spectrum of compound C 117 Figure 4.27 FAB- spectrum of compound C 117 Figure 4.28 COSY spectrum of compound C 118 Figure 4.29 HSQC spectrum of compound C 118 Figure 4.30 UV spectrum of compound D 120 Figure 4.31 IR spectrum of compound D 120 Figure 4.32 1H-NMR spectrum of compound D