Isolation and Structural Studies on Bioactive Constituents of Viscum album and Withania coagulans Thesis submitted for The Partial Fulfillment of the Degree of DOCTOR OF PHILOSOPHY (Chemistry) by SAIM A M AHE R H. E. J. Research Institute of Chemistry International Center for Chemical and Biological Sciences University of Karachi, Karachi-75270, Pakistan 2012 Dedicated To My Mother Whose inspiration and prayer always worked with me To My Father Whose teaching and blessings have only made this venture possible To My Husband Whose love, care and invaluable patronage are the most important assets of my life CERTIFICATE To Whom It May Concern It is certified that the thesis entitled, “Isolation and Structural Studies on Bioactive Constituents of Viscum album and Withania coagulans,’’ has been submitted by Mrs. Saima Maher to the Board of Advanced Studies and Research, University of Karachi, for the award of the degree of Doctor of Philosophy (Chemistry). She carried out her research under my supervision. The contents of this thesis, in full or in parts, have not been submitted to any other Institute or university for the award of any degree or diploma. PROF. DR. M. IQBAL CHOUDHARY Hilal-e-imtiaz, Sitara-e-imtiaz, Tamagha-e-imtiaz Director/Research Supervisor H. E. J. RESEARCH INSTITUTE OF CHEMISTRY International Center for Chemical and Biological Sciences University of Karachi, Karachi-75270, Pakistan CERTFICATE OF THESIS EVALUATION This is to certify that the Ph.D. thesis of Ms. Saima Maher d/o Mr Muhammad Israil,entitled Isolation and Structural Studies on Bioactive Constituents of Viscum album and Withania coagulan supervised by Prof. Dr. Prof. Dr. Muhammad Iqbal Choudhary H.I.,S.I.,T.I. has been checked and evaluated with positive comments by the following faculty members of the H.E.J Reasearch Institute of Chemistry. 1. Prof. Dr. M. Iqbal Choudhary H.I., S.I., T.I. 2. Prof. Dr. Viqar Uddin Ahmed H.I., S.I. 3. Prof. Dr. Bina Shaheen SiddiquiS.I.,T.I. 4. Prof. Dr. Khalid M. Khan T.I. International Center for Chemical and Biological Sciences, H. E. J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan Content List CONTENTS (i) ACKNOWLEDGEMENT i (ii) PERSONAL INTRODUCTION iv (iii) SUMMARY v (iv) URDU SUMMARY (Khulasa) x 1.0 GENERAL INTRODUCTION 1.1 INTRODUCTION 1 1.1.1 Medicinal Importance of Plants 1 1.1.2 History of Research on Medicinal Plants 2 1.1.3 Economic Value of Botanicals 3 1.1.4 Drug Discovery from Nature 4 1.1.5 Objectives of the Current Study 6 PART-A 2.0 PHYTOCHEMICAL STUDIES ON BIOACTIVE METABOLITES FROM VISCUM ALBUM L. 2.1 INTRODUCTION 8 2.1.1 The Family Loranthaceace 8 2.1.2 The Genus Viscum L. 7 2.1.3 Previous Phytochemical Investigation on Genus Viscum 9 2.1.4 The Plant Viscum album L. 14 2.1.5 Biological Significance of Viscum album L. 15 2.1.6 Significance of Constituents of Viscum album L. 15 2.1.7 Major Secondary Metabolites of the Plant: Flavonoids 16 2.1.7.1 Biosynthesis of Flavonoids 17 2.1.8 Minor Secondary Metabolites of the Plant: Phenylpropanoids 19 2.1.8.1 Biosynthesis of Phenylpropanoids 19 2.2 RESULTS AND DISCUSSION 22 2.2.1 Isolation of Major, and Minor Constituents from Viscum album 22 2.2.2 New Compounds 1 and 2 23 2.2.2.1 4′-O-[-D-Apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapyl-flavanone 23 (1) 2.2.2.2 3-(4′-Acetoxy-3′,5′-dimethoxy)phenyl-2E-propenyl-β-D-glucopyrnoside (2) 32 2.2.3 Known Compounds 3-10 39 Content List 2.2.3.1 3-(4′-Hydroxy-3′,5′-dimethoxy)phenyl-2E-propenyl-β-D-glucopyranoside (3) 39 2.2.3.2 5,7-Dimethoxy-4′-O-β–D-glucopyranosideflavanone (4) 40 2.2.3.3 5,7-Dimethoxy-4′-hydroxyflavanone (5) 41 2.2.3.4 4′,5-Dimethoxy-7-hydroxyflavanone (6) 42 2.2.3.5 7-Hydroxy-8methoxy flavanone (7) 43 2.2.3.6 Betuline (8) 45 2.2.3.7 Gallic Acid (9) 46 2.2.3.8 β-Sitosterol (10) 47 2.3 EXPERIMENTAL 48 2.3.1 General Experimental Conditions 48 2.3.1.1 Physical Constants 48 2.3.1.2 Spectroscopic Techniques 49 2.3.1.3 Purification and Detection of Compounds on Chromatographic Plates 49 2.3.2 Plant Material 49 2.3.3 Extraction and Fractionation of Methanol- Choloroform Soluble Constituents of Viscum 49 album 2.3.3.1 4′-O-[-D-Apiosyl(1→2)]--D-glucosyl]-5-hydroxyl-7-O-sinapyl-flavanone 52 (1) 2.3.3.2 3-(4′-Acetoxy-3′,5′-dimethoxy)-phenyl-2-(E)-propenyl-β-D-gluco-pyranoside 53 (2) 2.3.3.3 3-(4′-Hydroxy-3′,5′-dimethoxy)-phenyl-2E-propenyl-β-D-glucopyranoside 54 (3) 2.3.3.4 5,7-Dimethoxy-4′-O-β–D-glucopyranosideflavanone (4) 55 2.3.3.5 5,7-Dimethoxy-4′-hydroxyflavanone (5) 56 2.3.3.6 4′,5-Dimethoxy-7-hydroxyflavanone (6) 57 2.3.3.7 7,8-Dihydroxyflavanone (7) 58 2.3.3.8 Betuline (8) 59 2.3.3.9 Gallic Acid (9) 60 2.3.3.10 β-Sitosterol (10) 61 SECTION-A 3.0 BIOLOGICAL ACTIVITIES OF COMPOUNDS FROM VISCUM ALBUM L. 3.1 ANTIOXIDANT ACTIVITY 62 3.1.1 Introduction 62 3.1.2 Results and Discussion 63 Content List 3.1.3 Methodology 64 3.2 ANTI-GLYCATION ACTIVITY 65 3.2.1 Introduction 66 3.2.2 Results and Discussion 66 3.2.3 Methodology 68 PART-B 4.0 PHYTOCHEMICAL STUDIES ON SECONDARY METABOLITES FROM WITHANIA COAGULANS DUN. (STOCK.) 4.1 INTRODUCTION 71 4.1.1 The Family Solanaceae 71 4.1.2 The Genus Withania 71 4.1.3 Previous Phytochemical Studies on Genus Withania 72 4.1.4 Major Secondary Metabolites of the Plant: Withanolides 77 4.1.4.1 Biosynthesis of Withanolides 78 4.2 RESULTS AND DISCUSSION 83 4.2.1 Isolation of Major and Minor Constituents from Withania coagulans 83 4.2.2 New Compounds 11-12 84 4.2.2.1 (20R,22R)14,16α,17α,20α-Tetrahydroxy-1-oxo-witha-5,24-dienolide-3-O-β- 84 D-glucopyranoside (11) 4.2.2.2 3β,17β-Dihydroxy-14,20-epoxy-1-oxowitha-5,24-dienolide-27-O-β-D- 91 glucopyranoside (12) 4.2.3 Known Compounds 13 -17 98 4.2.3.1 Withanolid J (13) 98 4.2.3.2 Coagulin E (14) 99 4.2.3.3 Withaperuvin C (15) 100 4.2.3.4 27-Hydroxywithanolide I (16) 101 4.2.3.5 Ajugin E (17) 103 4.3 EXPERIMENTAL 104 4.3.1 General Experimental Conditions 104 4.3.1.1 Chromatography 104 4.3.1.2 Purification and Detection of Compounds on Chromatographic Plates 105 4.3.2 Isolation of Major and Minor Constituents from Withania coagulans 105 4.3.3 New Compounds 11 - 12 108 Content List 4.3.3.1 (20R,22R)14,16,17,20-Tetrahydroxy-1-oxo-witha-5,24-dienolide-3-O-β-D- 108 glucopyranoside (11) 4.3.3.2 3β,17β-Dihydroxy-14,20-epoxy-1-oxowitha-5,24-dienolide-27-O-β-D- 108 glucopyranoside (12) 4.3.3.3 Withanolid J (13) 109 4.3.3.4 Coagulin E (14) 110 4.3.3.5 Withaperuvin C (15) 111 4.3.3.6 27-Hydroxywithanolide I (16) 112 4.3.3.7 Ajugin E (17) 113 SECTION-B 5.0 BIOLOGICAL ACTIVITIES OF COMPOUNDS FROM WITHANIA COAGULANS DUN. (STOCK.) 5.1 CYTOXICITY ACTIVITY 117 5.1.1 Introduction 117 5.1.2 Results and Discussion 118 5.1.3 Methodology 118 6.0 REFERENCES 121 7.0 GLOSSARY 127 8.0 LIST OF PUBLICATIONS 1 30 Acknowledgement ACKNOWLEDGEMENT First of all, I am extremely grateful to the Almighty Allah (SWT), the most merciful and beneficent, for His unlimited guidance and blessings upon me to accomplish my goal. I pay my gratitude to Prof. Dr. Atta-ur-Rahman F.R.S., N.I.,H.I.,S.I.,T.I. for his dedicated efforts to equip, and modernize the H. E. J. Research Institute of Chemistry (International Center for Chemical and Biological Sciences). I wish to express my heartiest, and profound gratitudes to my dearest, lovely, highly respectable, kind hearted, ideal teacher, and supervisor Prof. Dr. Muhammad Iqbal Choudhary H.I.,S.I.,T.I. Director, ICCBS. His valuable gaudiness has brought success to my efforts. His impulsive, cheerful, endless, and harmonious encouragements lift my spirit, and raised my personality to meet the challenges. His intriguing, unique, and outstanding personality is a great motivator for me. I thank to Allah (SWT) and feel myself extremely fortunate that I am his student. He guided me and helped me a lot, and Inshaallah he will support me in future also. I got the lesson of self-respect from his personality. Whatever I was able to achieve, as presented here, is only due to his keen interest and support. Special expression to my Dearest Famiy members: I wish to express my respect to my family for their trust, prayers, and everlasting support. Really I have wonderful parents. Mrs. Mehrun Nisa, and Mr. Muhammad Israil. I can not do anything without their help, and support. I am thankful for their continuous encouragement and prayers for my success. I have no words to describe my true feelings for them. I am indebted to my dearest sisters, Mrs. Almas, Mrs.