PHYTOCHEMICAL STUDIES ON THE BIOACTIVE CONSTITUENTS OF HYPERICUM OBLONGIFOLIUM A thesis submitted to the University of the Punjab For the Award of Degree of Doctor of Philosophy in CHEMISTRY BY ANAM SAJID INSTITUTE OF CHEMISTRY UNIVERSITY OF THE PUNJAB LAHORE 2017 DEDICATION I Dedicate my work To my Parents My Husband And My Little Angels Ghanim and Afnan DECLARATION I, Anam Sajid d/o Sajid Saddique, solemnly declare that the thesis entitled “Phytochemical Studies on the Bioactive Constituents of Hypericum oblongifolium” has been submitted by me for the fulfillment of the requirement of the degree of Doctor of Philosophy in Chemistry at Institute of Chemistry, University of the Punjab, Lahore, under the supervision of Dr. Ejaz Ahmed and Dr. Ahsan Sharif. I also declare that the work is original unless otherwise referred or acknowledged and has never been submitted elsewhere for any other degree at any other institute. Anam Sajid Institute of Chemistry, University of the Punjab, Lahore APPROVAL CERTIFICATE It is hereby certified that this thesis is based on the results of experiments carried out by Ms. Anam Sajid and that it has not been previously presented for a higher degree elsewhere. She has done this research work under our supervision. Also we found no typographical and grammatical mistake while reviewing the thesis. She has fulfilled all requirements and is qualified to submit the accompanying thesis for the award of the degree of Doctor of Philosophy in Chemistry. Supervisors Dr. Ejaz Ahmed Institute of Chemistry, University of the Punjab, Lahore, Pakistan. Dr. Ahsan Sharif Institute of Chemistry, University of the Punjab, Lahore, Pakistan. Acknowledgement Saying of Prophet Muhammad (PBUH) ‘a person who is not thankful to his benefactors is not thankful to Allah’ so I would like to thank all the people who contributed in some way to the work described in this thesis. All and every kind of praises is upon ALLAH Almighty the strength of universe, who ever help in darkness and difficulties. I have a great honour to offer my heartiest gratitude to my worthy advisors Dr. Ejaz Ahmed & Dr. Ahsan Sharif for their inspiring guidance without which this work would never been materialized. Sir, you have been a tremendous mentor for me. I would like to thank you for encouraging my research and for allowing me to grow as a research scientist. Your advice on both research as well as on my career have been invaluable. I also want to thank you for letting my defense be an enjoyable moment, and for your brilliant comments and suggestions, thanks to you. Every result described in this thesis was accomplished with the help and support of HEC and HEJ so I greatly appreciate their cooperation as they responded promptly and enthusiastically to my requests despite their congested schedules. A special thanks to my husband Ghulam Mustafa, mother-in-law, father-in-law and mother, words cannot express how grateful I am to my family for all of the sacrifices that they’ve made on my behalf. I greatly benefited from suggestions of my beloved sisters Arfaa and Najam. Their prayer for me was what sustained me thus far. I would also like to thank to my fellows Faiza, Naila, Sumra, Fatima and Zahid. Thanks to Faiza and Naila for supporting me for everything, and especially I can’t thank you enough for encouraging me throughout this experience. To my beloved kids Ghanim and Afnan, I would like to express my thanks for being such good guys always cheering me up. Finally I thank my God, Allah Almighty, for letting me through all the difficulties. I have experienced His guidance day by day. He is the one who let me finish my degree. I will keep on trusting Him for my future. Anam Sajid TABLE OF CONTENTS Chapter 1 Introduction 1-16 1.1 Introduction 1 1.2 Terpenes 4 1.3 Steroids 7 1.4 Phenolics 8 1.5 Alkaloids 10 1.6 Hypericaceae family 13 1.7 Genus Hypericum 13 1.8 Hypericum oblongifolium 15 1.9 Pharmacological importance of H. oblongifolium 15 Chapter 2 Review of Literature 17-68 2.1 Review of Literature of Hypericum oblongifolium 17 2.1.1 Structures of the compounds previously isolated from 25 Hypericum oblongifolium 2.2 Biosynthesis of Terpenes 31 2.2.1 Biosynthetic route for terpenes 31 2.2.2 Cyclization of Squalene 36 2.2.3 Oxidative cyclization of squalene 38 2.2.3.1 Cyclization of squalene epoxide in chair-chair-chair-boat 38 sequence 2.2.3.2 Lupane and Hopane series 44 2.2.4 Non-oxidative cyclization of squalene 46 2.2.5 Cyclization at both ends of squalene molecule 47 2.3 Biosynthesis of steroids 48 2.3.1 Formation of Cholesterol from Lanosterol 49 2.3.2 Stigmasterol and β-Sitosterol 52 2.4 Biosynthesis of Fatty Acids 55 2.5 Biosynthesis of flavonoids 60 2.5.1 Biosynthesis of Flavanone 65 2.5.2 Formation of Isoflavone 66 2.5.3 Formation of flavone 67 2.5.4 Formation of Flavanol 68 Chapter 3 Experimental 69-109 3.1 General Experimental Conditions 69 3.1.1 Physical constants 69 3.1.2 Spectroscopy 69 3.1.3 Chromatography 70 3.1.4 Spray reagent for visualization of spot 71 3.1.4.1 Ceric Sulphate Reagent 71 3.2 Extraction and isolation 71 3.2.1 Plant material 71 3.2.2 Extraction and Isolation 71 3.3 Characterization of new compounds 77 3.3.1 Hyperinoate A (86) 77 3.3.2 Hyperinoate B (87) 79 3.3.3 Hyperinone (88) 81 3.3.4 Hyperinoic acid (89) 83 3.4 Characterization of known compounds 85 3.4.1 4,4-Dimethyl cholesterol (90) 85 3.4.2 β-Sitosterol (91) 87 3.4.3 Lupeol (92) 89 3.4.4 Taraxerol (93) 91 3.4.5 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (94) 93 3.4.6 Oleanolic acid (95) 95 3.4.7 Erectasteroid D (96) 97 3.4.8 (S)-4', 5-Dihydroxy-7-methoxyflavanone (97) 99 3.4.9 7, 4'-Dihydroxy-5, 3'-dimethoxyisoflavone (98) 101 3.4.10 α-D-Glucopyranosyl-6'-O-hexadecanoate (99) 103 3.4.11 β-sitosterol-3-O-β-D-glucopyranoside (100) 104 3.4.12 Quercetin-3'-O-β-D-glucopyranoside (101) 106 3.5 Biological screening 108 3.5.1 Lipoxygenase Inhibitory Assay 108 3.5.2 Antibacterial assay 108 3.5.3 Antifungal assay 108 Chapter 4 Results and Discussion 110-166 4.1 Hyperinoate A (86) 110 4.2 Hyperinoate B (87) 116 4.3 Hyperinone (88) 121 4.4 Hyperinoic acid (89) 127 4.5 4,4-Dimethyl cholesterol (90) 133 4.6 β-Sitosterol (91) 135 4.7 Lupeol (92) 136 4.8 Taraxerol (93) 138 4.9 4,4-Dimethylergosta-8,14,24(28)-triene-3β,12β,17α-triol (94) 140 4.10 Oleanolic acid (95) 142 4.11 Erectasteroid D (96) 144 4.12 (S)-4', 5-Dihydroxy-7-methoxyflavanone (97) 146 4.13 7, 4'-Dihydroxy-5, 3'-dimethoxyisoflavone (98) 148 4.14 α-D-Glucopyranosyl-6'-O-hexadecanoate (99) 150 4.15 β-sitosterol-3-O-β-D-glucopyranoside (100) 152 4.16 Quercetin-3'-O-β-D-glucopyranoside (101) 154 4.17 Biological screening 156 4.17.1 Lipoxygenase inhibitory activity 156 4.17.1.1 Lipoxygenases 156 4.17.1.2 The 5-Lipoxygenase pathway 156 4.17.1.3 Lipoxygenase inhibitory activity of compounds 86-101 159 4.17.2 Antimicrobial activity 161 4.17.2.1 Introduction 161 4.17.2.2 Antibacterial activity 161 4.17.2.3 Antifungal activity 162 4.17.2.4 Antimicrobial activities of compounds 86-101 164 Conclusion 167 References 168 LIST OF TABLES Table 1 Classification of Terpenes 4 Table 2 Classification of Phenolic compounds 8 Table 3 Classification of Alkaloids 12 Table 4 Compounds previously isolated from Hypericum 21 oblongifolium 1 13 Table 5 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 115 of compound 86, with J values (Hz) in parenthesis. 1 13 Table 6 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 120 of compound 87, with J values (Hz) in parenthesis. 1 13 Table 7 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 126 of compound 88, with J values (Hz) in parenthesis. 1 13 Table 8 H-NMR (500 MHz, CDCl3) C-NMR (125 MHz, CDCl3) 132 of compound 89, with J values (Hz) in parenthesis. Table 9 In vitro quantitative inhibition of lipoxygenase by 160 compounds 86-101. Table 10 Antibacterial and Antifungal activity of compounds 86-90 165 from Hypericum oblongifolium Table 11 Antibacterial and Antifungal activity of compounds 91-101 166 from Hypericum oblongifolium LIST OF FIGURES Figure 1 Structures of some Terpenoids 6 Figure 2 Structures of some common steroids 7 Figure 3 Structures of some common flavonoids 10 Figure 4 Structures of some common alkaloids 11 Figure 5 Different parts of Hypericum oblongifolium 16 Figure 6 Important HMBC correlations in 86 114 Figure 7 Important NOESY correlations in 86 114 Figure 8 Important HMBC correlations in 87 119 Figure 9 Important NOESY correlations in 87 119 Figure 10 Important HMBC correlations in 88 124 Figure 11 Important NOESY correlations in 88 125 Figure 12 Important HMBC correlations in 89 130 Figure 13 Important NOESY correlations in 89 131 Figure 14 The translocation of 5- Lipoxygenase, and cPLA2, to the nucleus, 157 upon cellular, stimulus following by the leukotrienes generation. Figure 15 Bacterial mode of action 162 LIST OF ABBREVIATIONS BB Broad Band CDCl3 Deutrated Chloroform 13C-NMR Carbon-13 nuclear magnetic resonance CoA Coenzyme A d Doublet dd Doublet of doublet DEPT Distortionless enhancement by Polarization transformation e.g.