MEDICINAL PLANTS OF NEPAL: Ethnomedicine, Pharmacology, and Phytochemistry by ROBIN S. L. TAYLOR B.Sc.H. Acadia University, 1992 A THESIS SUBMITTED IN PARTITAL FUFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Botany Program We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1996 ©Robin Taylor, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Bo^cvrx.^ The University of British Columbia Vancouver, Canada Date S<Lf>l • DE-6 (2/88) II Abstract Information about the medicinal uses of forty-two plant species was collected from traditional healers and knowledgeable villagers from a variety of different ethnic groups in Nepal. Illnesses for which these plants are used are those perceived in western style medicine to be caused by bacterial, fungal or viral pathogens. Methanol extracts of the species were screened for activity against a variety of bacteria, fungi and viruses, under various light conditions to test for photosensitizers. Thirty-seven extracts showed activity against bacteria and thirty-five showed activity against fungi. Only eight were active against Gram-negative bacteria. The exposure to UV-A light had a considerable effect on the activities of some extracts, with eight extracts being active only when exposed to light. The antibacterial and antifungal effects of fifteen extracts were enhanced upon exposure to light. Fifteen extracts showed 100% inactivation of at least one virus, and fifteen showed partial activity. Eight extracts were active only when exposed to light, and the antiviral effect of eight extracts was enhanced upon exposure to light. A species showing antibacterial activity, Centipeda minima (Asteraceae), and one showing antiviral activity, Carissa carandas (Apocynaceae) were the focus of bioactivity guided fractionation. Centipeda minima was found to contain three sesquiterpene lactones, identified as 6-O-methylacrylylplenolin, 6-O-isobutyroylplenolin, and 6-O-angeloylplenolin. 6-O-Methylacrylylplenolin had not been previously isolated from C. minima. All three of these sesquiterpene lactones had activity against Bacillus subtilis and Staphylococcus aureus. A fraction from the methanol extract of Carissa carandas was quite active against herpes simplex virus. This fraction was found to contain a derivative of 3,4,5-trimethoxycinnamic acid. Table of Contents Abstract ii Table of Contents iii List of Tables ix List of Figures xi Acknowledgements xiii Forward xiv Chapter I. General Introduction Ethnomedicine 1 Scope of Project 8 References 9 Chapter II. Ethnomedicine of Nepal Introduction 10 Plant Selection and Collection 15 Northern Nepal Plant Selection and Collection 16 Results 17 Discussion of the Northern Nepalese Collection. 27 Southern Nepal Plant Selection and Collection 28 Results 31 Discussion of the Southern Nepalese Collection. 43 General Discussion 44 References 49 Chapter III. Antimicrobial Screening Introduction 52 iv Antimicrobial screening of northern Nepalese medicinal plants Methodology Extract and Disc Preparation 59 Microorganisms 59 Antibacterial/Antifungal Assays 60 Results . 60 Discussion of Screening of Northern Nepalese Plant Extracts 66 Antimicrobial Screens of Southern Nepalese Medicinal Plants Methodology Extract and Disc Preparation 67 Microorganisms 67 Antibacterial/Antifungal Assays 67 Results 69 Discussion of Screening of Southern Nepalese Plant Extracts 74 General Discussion 75 References 79 Chapter IV. Antiviral screening Introduction 82 Methodology Extract Preparation 89 Cell Culture and Cytotoxicity Assays 89 Antiviral Assays 89 Results (Northern Nepalese Medicinal Plants) 95 Discussion (Northern Nepalese Medicinal Plants), 98 V Results (Southern Nepalese Medicinal Plants) 99 Discussion (Southern Nepalese Medicinal Plants) 103 General Discussion 104 References 108 Chapter V. Isolation and Identification of Several Bioactive Components from Centipeda minima Introduction 111 Methodology Antibacterial Bioassays 112 Plant Extract Preparation 114 Chemical Separations Liquid-Liquid Partition Chromatography 114 Column Liquid Chromatography 114 Thin Layer Chromatography 114 Vacuum Liquid Chromatography 115 High Performance Liquid Chromatography 115 Structural Analysis Ultraviolet Spectroscopy (UV-Vis) 115 Mass Spectroscopy (MS) 115 Nuclear Magnetic Resonance Spectroscopy 116 Results Bioactivity Guided Fractionation Liquid-Liquid Partitioning 116 Fractionation Procedures.... 119 vi Structural Identification Compound 1 Ultraviolet Spectroscopy 121 Mass Spectrometry 121 High Resolution Mass Spectrometery 121 Proton Nuclear Magnetic Resonance Spectroscopy 121 Carbon Nuclear Magnetic Resonance Spectroscopy 122 HMQC 122 Compound 2 Ultraviolet Spectroscopy 131 Mass Spectrometry 131 High Resolution Mass Spectrometery 131 Proton Nuclear Magnetic Resonance Spectroscopy 131 Carbon Nuclear Magnetic Resonance Spectroscopy 131 HMQC 131 Compound 3 Ultraviolet Spectroscopy 140 Mass Spectrometry 140 High Resolution Mass Spectrometery 140 Proton Nuclear Magnetic Resonance Spectroscopy 140 Carbon Nuclear Magnetic Resonance Spectroscopy 140 HMQC 140 HMBC 141 Antibacterial Activity 151 vii Discussion 153 References 159 Chapter VI. Isolation of an Antiviral Constituent of Carissa carandas Introduction 161 Methodology Antiviral Bioassays 162 Plant Extract Preparation 162 Chemical Separations Liquid-Liquid Partition Chromatography 163 Thin Layer Chromatography 163 Vacuum Liquid Chromatography 163 Preparative High Performance Liquid Chromatography 164 Analytical High Performance Liquid Chromatography 164 Structural Analysis Ultraviolet Spectroscopy (UV-Vis) 164 Mass Spectroscopy (MS) 164 Nuclear Magnetic Resonance Spectroscopy 165 Results Bioactivity Guided Fractionation Liquid-Liquid Partitioning 165 Fractionation Procedures 166 Structural Identification Compound 4 168 Discussion 178 References 183 vi Chapter VII. Summary Discussion 185 References 189 Appendix 1 190 Appendix 2 201 ix List of Tables 1.1 A partial list of commonly prescribed drugs that have been developed from plants, based on ethnobotanical information 4 2.1 List of northern Nepalese plants, their common names and medicinal use(s) 19 2.2 List of southern Nepalese plants, their common names and medicinal use(s) 32 3.1 A List of Common Hospital-Acquired (Nosocomial) Infections 54 3.2 Antibacterial results of northern Nepalese plant extracts 62 3.3 Antifungal results of northern Nepalese plant extracts 64 3.4 Antibacterial activities of southern Nepalese plant extracts 70 3.5 Antifungal activities of southern Nepalese plant extracts 72 4.1 Some Antiviral Phytochemicals, with source plant and activities 88 4.2 Cytotoxicty levels of the northern Nepalese plant extracts 91 4.3 Cytotoxicty levels of the southern Nepalese plant extracts 93 4.4 Northern Nepalese Plants: most active extracts 96 4.5 Northern Nepalese Plants: partially active extracts 97 4.6 Most active extracts of southern Nepalese plants 101 4.7 Partially active extracts of southern Nepalese plants 102 5.1 Activities of C. minima liquid-liquid partition fractions against Bacillus subtilis '. 117 5.2 1H-NMR assignments for 6-O-methylacrylylplenolin 127 5.3 13C-NMR assignments for 6-O-methylacrylylplenolin 129 5.4 HMQC data for 6-O-methylacrylylplenolin 130 5.5 1H-NMR assignments for 6-O-isobutyroylplenolin 136 5.6 13C-NMR assignments for 6-O-isobutyroylplenolin 138 X 5.7 HMQC data for 6-0-isobutyroylplenolin 139 5.8 1H-NMR assignments for 6-0-angeloylplenolin 146 5.9 13C-NMR assignments for 6-0-angeloylplenolin 148 5.10 HMQC data for 6-0-angeloylplenolin 149 5.11 HMBC data for 6-0-angeloylplenolin 150 5.12 Minimum inhibitory concentrations of the isolated sesquiterpene lactones 152 6.1 1H-NMR assignments for the active fraction of Carissa carandas 173 6.2 13C-NMR assignments for the active fraction of Carissa carandas ; 174 6.3 HMQC data for the active fraction of Carissa carandas 175 6.4 HMBC data for the active fraction of Carissa carandas 176 xi List of Figures 2.1 Map of Nepal 11 2.2 Lord Hanuman carrying a mountain 14 2.3 Collection areas in northern Nepal 18 2.4 Collection sites in sorthern Nepal 30 2.5 Gopal Shreshthe treating a young girl for 'side pains' 42 3.1 Disc assay of thiarubrine A against a variety of microorganisms in light and dark 58 4.1 Some synthetic antiviral drugs 86 4.2 Some antiviral phytochemicals 87 5.1 Activities of C. minima liquid-liquid partition fractions against Bacillus subtilis in dark and UV-A radiation 118 5.2 HPLC chromatogram of active components 120 5.3 Structure of 6-O-methylacrylylplenolin 123 5.4 UV spectrum of mixture containing 6-O-methylacrylylplenolin 124 5.5 El mass spectrum of mixture containing 6-O-methylacrylylplenolin 125 5.6 1H-NMR spectrum of mixture containing 6-O-methylacrylylplenolin , 126 5.7 13C-NMR spectrum of mixture containing 6-O-methylacrylylplenolin 128 5.8 Structure of 6-O-isobutyroylplenolin 132 5.9 UV spectrum of 6-O-isobutyroylplenolin 133 5.10 El mass spectrum of 6-O-isobutyroylplenolin 134 5.11 1 H-NMR spectrum of 6-O-isobutyroylplenolin