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Phytochemistry and biological activities of selected Colombian medicinal plants López Sarria, Andrés Augusto 2003

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PHYTOCHEMISTRY AND BIOLOGICAL ACTIVITIES OF SELECTED COLOMBIAN MEDICINAL PLANTS by A N D R E S A U G U S T O L O P E Z SARRIA B.Sc. (hons.), Universidad Nacional de Colombia, 1989 Diplome d'etudes approfondies (DEA) Universite de Paris Xll l 1993 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE FACULTY OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard The University of British Columbia January, 2003 © Andres Augusto Lopez Sarria, 2003 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 S>TrrAN v The University of British Columbia Vancouver, Canada Date / 3 / ZOO 3 DE-6 (2/88) ABSTRACT This thesis deals with the search for antibiotic compounds in medicinal plants traditionally used in the treatment of skin morbid conditions by First Nations People in Colombia. The plant collection was carried out in four culturally and geographically different regions of Colombia. Twenty-four chemical plant extracts were screened against different Gram positive and Gram negative bacteria, yeast and viruses. The plant extract from Piper lanceaefolium exhibited good activity against the yeast Candida albicans and the one from Iryanthera megistophylla displayed very good activity against herpes simplex virus and Staphylococcus aureus MS (methicillin-sensitive). Four compounds, not described in the literature, were isolated and further characterised from Piper lanceaefolium. Phytochemical analyses of Iryanthera megistophylla carried out by Dr. Dong Sheng Ming led to the isolation of two new compounds and seven known compounds. Amongst the known compounds a flavolignan, iryantherin K, presented potent activity against Staphylococcus aureus M S (methicillin-sensitive) and more importantly against Staphylococcus aureus MR (methicillin-resistant). Iryantherin K and its stereoisomer iryantherin L also exhibited good inhibition of the Candida albicans secreted aspartic protease, which is a virulent factor in Candida infections. The therapeutic potential that is offered by iryantherin K justifies a further toxicological and pharmacological assessment in animal models. ii TABLE OF CONTENTS Abstract ii Table of Contents iii List of Tables x List of Figures xii Acknowledgements \ xv Chapter 1 Introduction -1 1.1. Thesis Overview 1 1.2. Objectives 2 1.2.1. General Objective 2 1.2.2. Specific Objectives 2 1.3. Natural Products and Medicine: a Historical Perspective 2 1.3.1. Before Bayer 4 1.3.2. After Bayer 9 1.4. Search for Antibiotics in Medicinal Plants 11 1.5. Microbial Diseases of the Skin .14 1.5.1. Bacteria 14 1.5.2. Yeast. Candida albicans 15 1.5.3. Aspartic Proteases as Virulence Factors 18 1.5.4. Viruses 20 1.6. The Burden of Antibiotic Resistance 21 1.7. Culture and Environment i 26 1.7.1. Huitoto : - 2 6 1.7.2. Kamsa 27 1.7.3. Sikuani 28 1.7.4. Afro-Colombian Communities 29 1.8. References : 31 i i i Chapter 2 Field Collection of Colombian Medicinal Plants 40 2.1. Introduction 40 2.2. Materials and Methods 42 2.2.1. Plant Collection 42 2.2.2. Collection Sites 43 2.3. Results. Field Ethnomedical Data 47 2.4. Discussion 56 2.5. References • 59 Chapter 3 Biological Activities of Colombian Medicinal Plants 62 3.1. Introduction..... 62 3.2. Materials and Methods 64 3.2.1. Microorganisms 64 3.2.2. Preparation of Extracts 65 3.2.3. Antiviral Assays. Determination of Minimal Inhibitory Concentration (MIC) 65 3.2.4. Cytotoxicity Assays 67 3.2.5. Antimicrobial and Antifungal Assays 67 3.2.6. S A P Inhibition Screening 68 3.2.6.1. Induction of S A P s Production 68 3.2.6.2. Inhibitory Activity Against S A P s 69 3.2.6.2.1. Determination of Optimal Substrate and S A P Extract Concentrations 69 3.2.6.2.2. S A P Inhibition Assay for Crude Extracts 70 3.2.7. Electron Spin Resonance 71 3.3. Results 71 3.3.1. Antiviral Activity 71 3.3.2. Antimicrobial Activity 72 3.3.3. Inhibitory Activity Secreted Aspartic Proteases 73 i v 3.3.4. Inhibition of Secreted Aspartic Proteases 81 3.3.5. Electron Spin Resonance Spectroscopy for Juglans neotropica Extract..84 3.4. Discussion 87 3.5. Conclusion 90 3.6. References 91 C h a p t e r 4 Antifungal Activity of Benzoic Acid Derivatives from Piper lanceaefolium Kunth 95 4.1. Introduction..... 95 4.2. Materials and Methods 97 4.2.1. General Experimental Procedures 97 4.2.2. Plant Material 98 4.2.3. Extraction and Bioassay 98 4.2.4. Isolation of Compounds 100 4.2.5. Direct Bioautographic Assay 100 4.2.6. Minimum Inhibitory Concentration (MIC) Determination 101 4.2.7. Synergy Effects 102 4.2.8. Secreted Aspartic Proteases Inhibition Assay for Isolated Compounds 103 4.3. Results 103 4.3.1. Plant Description 103 4.3.2. Chemical Isolation and Characterisation ....104 4.3.3. Biological Activity .106 4.3.4. Aspartic Proteases Inhibition Assay 108 4.4. Discussion '. 110 4.4.1. Structural Elucidation 110 4.4.2. Biological Activity 117 4.5. Conclusions 122 4.6. References • ...123 v Chapter 5 Biological Activities from Iryanthera megistophylla A. C. Sm. Compounds 5.1. Introduction 128 5.2. Materials and Methods 130 5.2.1. Anti-viral Assays. Determination of Minimal Inhibitory Concentration (MIC)] 130 5.2.2. Cytotoxicity Assay.. 130 5.2.2.1. Cytotoxicity Visible Assessment 130 5.2.2.2. Cell Proliferation Assay for Iryantherin K 131 5.2.3. Antibacterial and Antifungal Assays 131 5.2.3.1. Minimum Inhibitory Concentration (MIC) Determination 131 5.2.3.2. Test for Multidrug Resistance (MDR) Inhibitory Activity 131 5.2.4. Secreted Aspartic Proteases Inhibition Assay 132 5.2.5. Pepsin Inhibition Assay 132 5.3. Results 132 5.3.1. Cytotoxicity 132 5.3.2. Antiviral and Antimicrobial Activities 133 5.3.3. Secreted Aspartic Proteases Inhibition 140 5.3.4. Inhibitory Activity of Iryantherin K (4) and L (5) 141 5.4. Discussion 143 5.5. Conclusion 148 5.6. References 149 Chapter 6 Conclusions 155 6.1. Search for Antibiotics in Medicinal Plants 155 6.2. Understanding Other's Medical Practices 159 6.3. References 163 v i Appendix 1 A. Nuclear Magnetic Resonance Spectrum of Cyclolanceaefolic acid methyl ester (1H) 400 MHz (1) 16 Appendix 1B. Nuclear Magnetic Resonance Spectrum of Cyclolanceaefolic acid methyl ester ( 1 3 C) 100 MHz (1) 166 Appendix 1C. Two-Dimensional ( 1H 1 H) Nuclear Magnetic Resonance Spectrum (COSY) of Cyclolanceaefolic acid methyl ester (400 MHz) (1) 167 Appendix 1D. Low Resolution El Mass Spectrum of Cyclolanceaefolic acid methyl ester (1) 168 Appendix 1E. High Resolution El Mass Spectrum of Cyclolanceaefolic acid methyl ester (1) 169 Appendix 1F. Infrared Spectrum of Cyclolanceaefolic acid methyl ester 170 Appendix 2A. Nuclear Magnetic Resonance Spectrum ( 1H) of Cyclolanceaefolic acid (2) (400 MHz) 171 Appendix 2B. Nuclear Magnetic Resonance Spectrum ( 1 3 C) of Cyclolanceaefolic acid (2) (100 MHz) 172 Appendix 2C. Low Resolution El Mass Spectrum of Cyclolanceaefolic acid (2) 17^ v i i Appendix 2D. High Resolution El Mass Spectrum of Cyclolanceaefolic acid (2) 174 Appendix 3A. Nuclear Magnetic Resonance Spectrum ( 1H) of Lanceaefolic acid methyl ester (3) (400 MHz) 175 Appendix 3B. Nuclear Magnetic Resonance Spectrum ( C) of Lanceaefolic acid methyl ester (3) (100 MHz) 176 Appendix 3C. Low Resolution El Mass Spectrum of Lanceaefolic acid methyl ester (3) 177 Appendix 3D. High Resolution El Mass Spectrum of Lanceaefolic acid methyl ester (3) 178 Appendix 4A. Nuclear Magnetic Resonance Spectrum ( 1H) of Lanceaefolic acid (4) (400 MHz) 179 Appendix 4B. Low Resolution El Mass Spectrum of Lanceaefolic acid (4) 180 Appendix 4C. High Resolution E l Mass Spectrum of Lanceaefolic acid (4) 181 Appendix 5A. Nuclear Magnetic Resonance Spectrum of Taboganic acid (5) (1H) 400 MHz 182 vi i i Appendix 5B. Low Resolution El Mass Spectrum of Taboganic acid 183 Appendix 6A. Nuclear Magnetic Resonance Spectrum of Pinocembrin (6) (1H) 400 MHz 184 Appendix 6B. Nuclear Magnetic Resonance Spectrum of Pinocembrin (6) ( 1 3 C) 100 MHz 185 Appendix 6C. Low Resolution El Mass Spectrum of Pinocembrin (6) 186 Appendix 7A. Nuclear Magnetic Resonance Spectrum of Pinocembrin chalcone (7) ( 1H) 400 MHz 187 Appendix 7B. Low Resolution El Mass Spectrum of Pinocembrin chalcone (7) (1H) 400 MHz 188 ix LIST OF TABLES Table 3.1. Antiviral Activities of Extracts of Selected Colombian Medicinal Plants 74 Table 3.2. Antimicrobial Activities of Extracts of Selected Colombian Medicinal Plants 77 \ \ \ Table 3.3. Inhibition of Secreted Aspartic Proteases by Colombian Medicinal Plant Extracts 82 Table 3.4 Hyperfine Splitting Constants (a values) (in Gauss) and g values of Juglone found in Juglans neotropica extract 86 Table 4.1 Microtiter Plate Assay to Test for Synergy Effects 102 Table 4.2 Inhibition of Secreted Aspartic Proteases by compounds from P. lanceaefolium 108 Table 4.3. 1 H and 1 3 C Data for Compounds 1-4 a- 109 Table 5.1 Minimal Inhibitory Concentrations of /. megistophylla compounds against herpes simplex virus, C. albicans ,S. aureus MS (methicillin-sensitive) and S. aureus MR (methicillin-resistant)... 135 i Table 5.2 Minimal Inhibitory Concentrations (pg/mL) of Iryantherin K Against Gram positive Bacteria 136 Table 5.3. Minimal Inhibitory Concentrations (pg/mL) of Iryantherin K Against Gram negative Bacteria 136 x Table 5.4. Secreted Aspartic Proteases Inhibition Activity of Compounds from /. megistophylla 140 Table 5.5. Inhibitory Activity of Iryantherin K and L against Secreted Aspartic Proteases (SAP's) by C. albicans 142 x i LIST OF FIGURES Figure 1.1. Gram-positive Bacterial Multidrug Pump 22 Figure 1.2. Structure of Berberine 24 Figure 1.3. Structure of 5'-methoxyhydnocarpin D (5' - M H C ) 4H-1-Benzopyran-4\one 25 Figure 2.1. Collection Sites in Colombia of the plants studied.... 44 Figure 3.1. E S R Semiquinone Spectrum of Juglone in Juglans neotropica Alcoholic Extract 85 Figure 4.1. Map of the Sibundoy Valley (Colombia) 99 Figure 4.2. Bio-autography with Candida albicans 107 Figure 4.3. Cyclolanceaefolic acid methyl ester (1) 111 Figure 4.4. Cyclolanceaefolic acid (2) 112 Figure 4.5. Lanceaefolic acid methyl ester (3) 113 Figure 4.6 Selected HMBC correlations for 1 and 3 114 i Figure 4.7 Lanceaefolic acid (4) 115 Figure 4.8. Taboganic acid (5) 3-(1 '-Oxo-3'-methyl-2'-butenyl)-4-hydroxy-benzoicacid 115 x i i Figure 4.9 Pinocembrin (5,7-Dihydroxyflavanone) (6) 116 Figure 4.10 Pinocembrin chalcone (7) 116 Figure 4.11 Chalcones (1,3-diaryl-2-propen1-ones) general skeleton 117 Figure 4.12 Effect of 2' Hydroxyl Group Removal in Chalcones Antifungal Activity (Tsuchiya, 1994) 118 Figure 4.13 Effect on Antifungal Activity of the Removal of 2'-Hydroxyl (Sato, 1994) 119 Figure 4.14 Asebogenin (2', 6', 4 - trihydroxy - 4 - methoxydihydrochalcone) 121 Figure 5.1 Structure of Cinchonain lb (6) 133 Figure 5.2 Structure of Cinchonain la (7) 133 Figure 5.3 Procyanidin B 2 o r catechin - (4p - 8) epicatechin (8) 134 Figure 5.4 Structure of Cinchonain Ha (9) 134 Figure 5.5 Iryantherin K (4) 137 Figure 5.6 Iryantherin L (5) ' 138 Figure 5.7 Megislignan (1) [2,3-dimethyl-4-(4-methoxyphenyl)-6-hydroxynaphthalene] 139 x i i i Figure 5.8 Megislactone (2) pR .SR^RJ -S -hyd roxy^ -me thy l ^ ^hexacos - l 7-enyl) butanolide] 139 Figure 5.9 Grandinolide (3) 140 x i v ACKNOWLEDGEMENTS I would like to thank Professor G. H. Neil Towers, for his encouragement and support during the course of this thesis and Professor Jim Hudson for introducing me to the fascinating field of virology and cell biology. His critical comments were very important in the making of this manuscript. I would also like \ to thank Professor lain Taylor for his advice and for his editorial comments in the writing of this thesis and Professor Bruce A. Bohm for generously sharing his expertise in natural products and allowing me to work in his laboratory for a long period of time. I am grateful to Dr. Dong Sheng Ming who helped me during the phytochemical separation and interpretation of the spectroscopic data. I would like to thank also Rene Orozco and Felipe Balza for sharing their expertise in phyto-chemistry and Zyta Abramowski for providing assistance during the antimicrobial tests. I thank Dr. R.E.W. Hancock and Susan Farmer (Department of Microbiology and Immunology, UBC) for helping me with some of the antimicrobial tests and Dr. Brett Finlay (Biotechnology Laboratory UBC) who kindly allowed me to use the fluorescence reader as well as Michelle Zaharik for providing assistance in its use. i I thank Dr. Michel Roberge (Department of Biochemistry and Molecular Biology) and Tamsin Tarling for helping me with the cell proliferation assay of iryantherin K. x v In Colombia, my sincerest thanks go to Don Miguel Chindoy and Dona Clarita Buesaquillo the couple of healers from the Kamsa community, Sibundoy Valley. Not only did they help me during the plant collection but also were generous in sharing their knowledge about medicinal plants and introducing me to the yage teachings. My immense gratitude to Hubert Murillo and his father Adriano Murillo in Bajo Calima for sharing their herbal medicine expertise and helping with the plant collection. The plant they pointed out is a major protagonist in this thesis and one of its compounds holds a promise in the fight against resistant bacteria. I would also like to thank Juan Alvaro Echeverri (National University-Leticia, Colombia) for his expertise in anthropology, his great sense of humour and tea at five in the middle of the Amazon rainforest, Emilce Mora (Etno-llano) for providing assistance during the plant collection in the Mataven forest and Dayron Cardenas and Alvaro Mogollon from the Sinchi Institute (Santa Fe de Bogota, Colombia) who helped me in the identification of the plant material. I would also like to thank Yolanda Sarria for her unconditional support, Juan Claudio Lopez for his valuable help in the preparation of plant material and Gloria Bernal de Sarmiento for her hospitality and for the uncomfortable task of bringing some of this plant material into Canada. Bernard Weniger (University of Strasbourg) played a major role in facilitating the collection of plant material in Bajo Calima and kindly lodged me during my stay in Cali . To him and his family my deepest acknowledgements. x v i I would also like to Dr. G. K. Eigendorf, Marietta Austria, and Liane Darge from the University of British Columbia for the acquisition of mass and NMR spectral data, Trevor Hayton (Department of Chemistry) and David E. Williams (Department of Oceanography) at UBC for the acquisition of the infrared spectra and the optical activity determinations respectively. The Natural Sciences and Engineering Research Council of Canada financially supported this research in the form of operating grants to Professor G.H.N. Towers. The financial support provided by Carlos Sarmiento played a very important role and certainly helped me in accomplishing this work. Finally I would like to express my sincerest thanks to my partner Monica Sarmiento. Her commitment and willingness to help in whatever I needed played a major role in the making of this thesis. She always had a word of encouragement in times when disappointment and frustration were frequent feelings during the thesis work. Her kindness always reminded me there was a goal to be achieved and that I was not by myself. Her comments and questions about my research over sushi or coffee (at the Pendulum) definitely enriched my perspectives in the field. I am most grateful to her. x v i i This thesis is dedicated to Eustorgio Sarria. Chapter 1 Introduction 1.1. Thesis Overview The search for chemical substances produced by plants, which are biocidal to viruses, yeast, and bacteria is the main focus of this research. A consideration of the traditional uses of a plant would seem to be a sensible approach towards this aim. This manuscript has been divided into six sections: the first section briefly introduces the historical context of natural medicines and presents information on the indigenous cultures I worked with. The second chapter deals with the field collection of selected Colombian medicinal plants traditionally used in the treatment of skin morbid conditions. The third chapter deals with the screening of plant chemical extracts against a selected battery of relevant strains of bacteria, fungi and viruses in order to determine biologically active extracts that merited further phyto-chemical analysis. Plant extracts of Piper lanceaefolium and Iryanthera megistophylla were found to present inhibitory activities against the yeast Candida albicans and the herpes simplex virus respectively. The fourth chapter describes the separation, isolation, purification and structural elucidation of seven compounds, from an acetone extract of P. i lanceaefolium, of which four are new to the literature and two were biologically active against Candida albicans. The fifth chapter includes a collaborative effort with the post-doctoral fellow Dong Sheng Ming. In this chapter the biological testing of isolated 1 compounds from Iryanthera megistophylla against herpes simplex virus and Staphyloccus aureus is described along with an enzymatic assay in order to assess the inhibitory activity of aspartic proteases which are considered a virulence factor in Candida infections. The final chapter encompasses a general discussion of the entire research and suggests future possible directions in the field. 1.2. Objectives 1.2.1. General Objective Search and identification of chemical compounds with biocidal and/or antiviral activities present in traditional medicinal plants from Colombia. 1.2.2. Specific Objectives > Collect and identify traditional medicinal plants from different cultural and geographical areas of Colombia. > Assess biological activities against relevant gram-positive and gram-negative bacteria, fungi and viruses. > Isolate and identify the chemical compounds that individually, or in conjunction with others, are responsible for such biocidal and/or antiviral activity. 1.3. Natural Products and Medicine: a Historical Perspective. Speaking of the origin of plants as medicinal agents I have always wondered about the number of unfortunate people who had to die or had a miserable time in their efforts to find a suitable cure for a given ailment. Keen 2 observation of animals' food consumption behaviour probably provided interesting clues in this regard. A discussion about the origins of medicine and its relationship with natural products is well beyond the scope of this introduction therefore, I will only mention selected historical landmarks of written records regarding the use of plants as medicines. Chemical ecologists have suggested that the human use of medicinal plants is related to the need for ingestion of plant secondary compounds as a defence mechanism against various parasitic microorganisms. Janzen (1978) suggested that mammals deliberately ingest plants to get rid of intestinal parasites. Antiparasiticity appears to be the primary non-nutrient function that has evolved in animal interactions with plant chemicals. It has been argued that control of parasitic and infectious diseases through the use of plants may have played a role in human plant selection (Johns, 1990). The way humans have articulated plant medicinal uses in a system of beliefs about nature is an issue that likely will never be resolved. However, I would think that the use of hallucinogenic plant preparations has likely played an important role in this regard. I will divide this short historical account in two parts. I will use Bayer's semi-synthesis of aspirin as a division because I consider this event a cultural landmark in the sense that it divests the plant or mixture of plants from their intrinsic value to be simply converted to a reservoir of single chemical entities. The notion of plants as resources of different kinds is at the very base of today's struggle over land use in developing and developed countries alike. 3 1.3.1. Before Bayer The first written records of traditional medicine systems are from Mesopotamia, written on hundreds of clay tablets in cuneiform, and date from about 2600 BC . However, it is important to mention they did not place importance on herbs. The herbal remedies of Ancient Egypt are described in exceptional detail by the best known Egyptian pharmaceutical record: the "Ebers Papyrus" dating from 1550 BC. Over 800 remedies are described in it, involving the use of plant extracts, animal organs and minerals. The Assyrians, who ruled in Mesopotamia in the first half of the first millennium B C , greatly consolidated ancient knowledge about herbal remedies. They left a legacy of 1500 years of herbal medicine (c 1900 to 400 BC) that extolled the virtues of around 1000 medicinal plants and remedies including enemas and poultices for exorcism of spirits (Mann, 2000). The Chinese Materia Medica has been extensively documented over the centuries with the first record dating from about 1100 BC. However, the first herbalist that concentrated on cataloguing and describing therapeutically effective substances appears to have been compiled in China by Shennong Bencao Jing (100-200 A D) and covered 365 herbal drugs at the same time as the materia medica of Dioscorides (Tang, 1992, Unschuld, 1988). In Chinese herbal medicine formulas are chosen on the basis of patterns of illness or imbalance, not just symptoms. This pharmacopoeia contains almost 6000 herbs usually formulated in mixtures of up to 20 herbs (Simpson, 2001). 4 Ayurvedic medicine is an old tradition from India and because it literally means "knowledge of life" encompasses more than just medicinal aspects, as it includes psychological, cultural, religious and philosophical concepts. Much of the knowledge is included in the Vedas books of knowledge and in Sambitas which contain the science of Ayurveda. This system classifies individuals according to three determined body types or doshas where the medicinal approach is to restore the balance of the whole body rather than suppress symptoms. Documentation of the Indian Ayurvedic system dates from about 1000 B C and provided the basis for the primary text of Tibetan medicine (Newman et al., 2000). A number of Ayurvedic herbs have become more widely known in the West including Justicia adhatoda (Acanthaceae) which is effective in respiratory conditions and Phyllanthus amarus (Euphorbiaceae) with its beneficial effects in liver disease (Sandberg and Corrigan, 2001). The concept of hot and cold medicines forms a cornerstone of the Humoral theory that originated in antiquity and developed out of the writings of Hippocrates and Aristotle. Hippocrates maintained that a person's health depended upon a delicate balance of four body humours, blood, phlegm, black bile, and yellow bile, and that an imbalance of these produced illness. This theory of disease, later refined by Galen, was the dominant medical ideology throughout the Renaissance (Furst, 1995). Galen believed that epidemics were caused by "miasmas" or poisonous vapours produced whenever the conjunction of the planets was unfavourable (Mann, 2000). 5 After the decline of the Greek empire with the death of Alexander the Great (323 BC), several contributions were accomplished and it is worth mentioning the eight volumes of De Medicina by Celsus that includes over 250 plant-derived remedies. Probably the most significant contribution was made by Dioscorides, a Greek physician (100 AD), during his travels with Roman armies. He recorded the collection,^storage, and use of medicinal herbs. His legacy was a five-volume work entitled De Materia Medica written in the first century A. D.; it described 600 plants and plant products. Although poorly organised it became the model for future pharmacopoeias (Simpson, 2001). Through the so-called "Dark Ages" in Europe, herbal knowledge was kept alive initially through the work of scribes in Constantinople and then in the libraries of the rapidly expanding Arab Empire. The Arabs were responsible for the preservation of much of the Greco-Roman expertise and for expanding it to include the use of their own resources together with Chinese and Indian herbs. The Mansurian Book of Medicine written by Rhazes (c. 865-925 A D) represents a major contribution to pharmacy. Rhazes demonstrated the toxicity of many of the popular remedies based on heavy metal salts, particularly those involving mercury (Mann, 2000). The contribution of Avicenna through works such as Canon Medicinae is regarded as "the final codification of all Greco-Roman medicine". This was subsequently superseded by the comprehensive compilation known as Corpus of Simples by Ibn al-Baytar (1197-1248) who practised in Malaga during the Moorish occupation of Spain. This treatise describes many items including 800 plant-derived drugs, 145 from minerals, and 130 from 6 animals. Also from the fifth to the twelfth centuries, monks in monasteries in Europe preserved the remnants of this Greco-Roman tradition. Paracelsus (1493-1541) believed that God placed plants on the Earth for human use. Consequently, God had provided signs embodied in the plants to indicate their potential uses. For instance if a plant had red sap, that was a sign that the plant was intended for the treatment of blood disorders. This idea was called the doctrine of signatures and is believed to have directed healers to the potential therapeutic benefits of the vegetation. He was a key figure in the Renaissance and also a pioneer chemist. With his emphasis on the medicinal use of substances such as antimony, arsenic, iron, sulfur and mercury, he laid the foundation of chemical pharmacology (Johns, 1990). Regarding the American continent, the oldest known medical text is the Badianus manuscript composed by Aztec scholars. It depicts many of the most important plants employed in Aztec medicine and it is the earliest pharmacopoeia we have from the Americas (Furst, 1995). The number of species employed by Native Americans is staggering. Moerman's two-volume compendium, Medicinal Plants of Native America (1998) has 17,000 entries. Unfortunately there is no comparable list for Middle and South America but considering the cultural and natural biodiversity particularly in Central America and in the Amazonian rain forest, the number may well be substantially higher (Furst, 1995). Indigenous people of Mesoamerica had, and still have, extensive knowledge of healing plants. 7 Conquest and colonisation of the Americas by Europeans were accompanied by an unprecedented blending of Old and New World diseases, ethnomedical systems, and plant-based pharmacopoeias (Voeks, 1993). The demographics of the slave trade most clearly delimited where and to what degree African magic-medical systems diffused into the New World. Also it has been argued that the religious division between Protestantism in British North America and Roman Catholicism in Latin America played a major role to the degree of cultural retention. Roman Catholic liturgy had some structural similarities with African religions, polytheism amongst them. According to Ribeiro cited in Voeks (1993) the conversion to Catholicism was best performed not by destroying their icons but rather by slowly replacing them with Roman Catholic symbols and rituals. Although it is not possible to generalise an African ethnomedical system, the idea of illness as a reaction to forces outside the realm of secular comprehension is a common feature. Shaman healers act as intermediates between the material and spiritual universes, and they seek out the otherworld sources of physical and emotional distress. In The Skeptical Chymist published in 1661 Robert Boyle laid the foundations for an understanding of the chemistry of the drugs. The discovery of i the effectiveness of Digitalis by William Withering (1741-1799) for the treatment of dropsy is considered by many as the beginning of modern pharmacology. The old ideas of Aristotle concerning the four elements (earth, air, fire and water) 8 were swept away and a chemical element was defined as a substance that could not be broken down into simpler substances (Mann, 2000). 1 . 3 . 2 . A f t e r B a y e r In the early 1800's the isolation of the active principles of commonly used plants and herbs such as strychnine from seeds of Nux vomica (1817), morphine (1816), atropine (1819), quinine (1820) and colchicine (1820) were achieved. The first semi-synthetic pure drug based on a natural product, acetylsalicylic acid (aspirin), by Bayer in 1899 then followed these isolations. In the first half of the twentieth century many substances with current medicinal use were obtained from traditional plant-derived extracts (Mann, 2000). Despite the success of single compound drugs in the Western world, it is estimated that over 80% of the world's population still relies on traditional medicines including herbal medicine, for primary health care (Farnsworth, 1985). Regardless of the,successful entries of organic synthesis in the post-world war II years, the need for structurally novel therapeutic categories continues and so do bio-prospecting projects world wide on behalf of major pharmaceutical companies. Ancient medical systems such as Chinese Traditional Medicine and the Ayurvedic system are alive despite the efforts of the pharmaceutical companies to dismiss them claiming health concerns. A s a matter of fact, traditional knowledge along with its associated pharmacopoeias has played a major role in drug discovery and it is a matter of political debate between native people and 9 p h a r m a c e u t i c a l c o m p a n i e s . It is e s t i m a t e d that at l e a s t 8 8 d r u g s a r e d e r i v e d f r o m p l an t s t rad i t i ona l l y u s e d . C o m p o u n d s s u c h a s v i n c r i s t i ne , v i n b l a s t i n e , t a xo l a n d t he f am i l y of a n t i m a l a r i a l s d e r i v e d f r o m q u i n i n e a n d a r t e m i s i n i n a r e o f g rea t i m p o r t a n c e a n d r e s p o n s i b l e for s a v i n g l i ve s w o r l d w i d e ( F a r n s w o r t h 1990) . A g o o d i l lust rat ion of the i n te rp lay b e t w e e n t r ad i t i ona l m e d i c i n e a n d d rug d i s c o v e r y i s t h e c a s e o f a m e m b e r in t he A p o c y n a c e a e f ami l y , Rauvolfia serpentina. H i n d u s a p p l i e d the n a m e Chandra ( m o o n ) to th i s p l an t s i n c e it w a s u s e d to t reat " m o o n d i s e a s e " o r l unacy . Ind ian s a d h u s h a v e c h e w e d on s n a k e r o o t s of Rauvolfia serpentina fo r c e n t u r i e s b e c a u s e o f its c a l m i n g e f f e c t s a n d a s a c o a d j u v a n t in r e a c h i n g " sp i r i t ua l e n l i g h tmen t " . It i s a l s o u s e d in India a n d pa r t i cu la r l y in the B i h a r p r o v i n c e to t reat i n san i t y , e p i l e p s y , a n d i n s o m n i a h e n c e the l o c a l n a m e pagal-ka-dawa " i n s an i t y c u r e " . In 1 9 4 9 E m i l Sch l i t t l e r , a c h e m i s t at C I B A p h a r m a c e u t i c a l s in B a s e l , S w i t z e r l a n d , e x t r a c t e d f r o m Rauvolfia roo t s a n a l k a l o i d a n d ve r i f i ed its d r a m a t i c p r o p e r t i e s to l o w e r t he b l o o d p r e s s u r e in a g r e e m e n t w i th p r e v i o u s repo r t s . T h i s c o m p o u n d w a s n a m e d r e s e r p i n e a n d b e c a m e t he f irst m a j o r d r u g to t reat o n e of t he m o s t s e r i o u s i l l n e s s e s of the W e s t e r n w o r l d : h y p e r t e n s i o n ( Ba l i c k a n d C o x , 1996 ) . T h e r e a l i s a t i o n that " p r im i t i ve s o c i e t i e s " st i l l h a d s o m e t h i n g to o f f e r to the w e s t e r n w o r l d p r o m p t e d a d v e n t u r e r s , t r a ve l l e r s , a n d s c i e n t i s t s f r o m d i f fe rent pa r t s of t he w o r l d to s e e k out h e a l e r s a n d s h a m a n s in a n a t t e m p t to u n c o v e r the i r k n o w l e d g e a b o u t p l an t s a n d the i r c u r a t i v e p r ope r t i e s . F o r a f a s c i n a t i n g a c c o u n t o f t h e s e e n t e r p r i s e s in C o l o m b i a , t he r e a d e r i s r e f e r r ed to W a d e D a v i s ' b o o k " O n e R i ve r " . 10 Today isolated natural products have become successful pharmaceuticals in the Western world. In a study done in the United States, from January through September of 1993, the top 150 proprietary drugs prescribed were analyzed and their origin determined. Amongst them, 18% of the top 150 most prescribed drugs have their origin in the kingdom Plantae (Grifo et al., 1997). Numerous scholars\have praised the wealth of knowledge of traditional societies and a note of urgency has been sent to rescue and document this body of knowledge. Indeed, Holmstedt and Bruhn (1995) declare, in regard to the "discipline" of ethno-pharmacology: "the objectives of ethno-pharmacology are to rescue and document an important cultural heritage before it is lost, and to investigate and evaluate the agents employed. " Traditional systems are considered as a collection of items to be investigated with no agency whatsoever. This statement implies the incapacity of non-western cultures in evolving and re-creating themselves in different cultural settings. It would appear that the purpose of the discipline is to obtain information out of a dying culture before it is too late. 1.4. Search for Antibiotics in Medicinal Plants Henceforth I will use the term "antibiotic" in a pharmacological sense exclusively and I will refrain from implying that such activity plays a role in nature. Historically, the search for clinically useful antibiotics in plants has been a failure. Until now, there is not a single plant natural product in the pharmaceutical antibiotic armamentarium. However, scientific literature is rich in information 11 a b o u t p l an t e x t r a c t s and/or p h y t o - c h e m i c a l s wi th b i o c i d a l p r o p e r t i e s a g a i n s t m i c r o b e s (e.g. v i r u s e s , b a c t e r i a a n d fung i ) that o f ten t i m e s p o s s e s s u n d e s i r a b l e t ox i c o r p h a r m a c o l o g i c a l p r o p e r t i e s p r e v e n t i n g t h e m f r o m b e c o m i n g u se fu l . C o w a n ( 1999 ) h a s r e cen t l y r e v i e w e d a n t i m i c r o b i a l s of p l an t o r i g i n a n d H u d s o n (1990 ) h a s c o m p i l e d a b o o k of ant i v i ra l c o m p o u n d s f r o m p l an t s . M o s t of the na tu ra l p r o d u c t s c l i n i ca l l y r e l e van t a s an t i b i o t i c s a r e b a s e d o n m e t a b o l i t e s f o u n d in m i c r o o r g a n i s m s (for a r e v i e w s e e N e w m a n et al., 2 000 ) . A su rp r i s i n g l y l im i ted g r o u p of o r g a n i s m s a r e s o u r c e s of m a n y an t i b i o t i c s in u s e today , m o r e t han ha l f of the an t i b i o t i c s cu r ren t l y u s e d a r e p r o d u c e d by s p e c i e s Streptomyces, f i l a m e n t o u s b a c t e r i a that c o m m o n l y i nhab i t s o i l ( To r t o r a , 2 0 0 2 ) . T w o c o m p o u n d s i s o l a t e d f r o m m a r i n e s p o n g e s : s p o n g o u r i d i n e a n d s p o n g o t h y m i d i n e , b e c a m e t he p ro to t ype fo r a s e r i e s o f a v a s t n u m b e r of d e r i v a t i v e s (i. e. a c y c l o v i r ) tha t w e r e t e s t e d e x t e n s i v e l y a s an t i v i r a l a n d an t i - t umo r a g e n t s a n d a r e n o w the cu r r en t ant i v i ra l t he r apy . F r o m a n e v o l u t i o n a r y p e r s p e c t i v e f a s c i n a t i n g p r o p o s a l s h a v e b e e n put f o r w a r d in r e g a r d s to t he ro le of an t ib i o t i c s . D a v i e s ( 1 990 ) h a s s u g g e s t e d that l o w - m o l e c u l a r w e i g h t s e c o n d a r y m e t a b o l i t e s h a v e p l a y e d a u n i q u e b i o c h e m i c a l ro le in e vo l u t i o n b e f o r e p r o te i n s w e r e a v a i l a b l e , a s m a n y b i o s y n t h e t i c r e a c t i o n s w e r e " c a t a l y s e d " by l ow m o l e c u l a r - w e i g h t m o l e c u l e s p r e s e n t in " p r i m o r d i a l s o u p r eac t i on s " . W h e t h e r an t i b i o t i c ac t i v i t i e s f o u n d in t he l a b o r a t o r y c o r r e l a t e to the t rad i t i ona l u s e is a m a t t e r of d e b a t e , a n d o n e m u s t e x e r t c a u t i o n in a d v a n c i n g 12 hypotheses that sometimes reflect scientist's cultural biases (e.g. euro-centrism) rather than scientific attributes. The principle that should be accepted is that the possession of any type of biological activity should not in itself be sufficient reason to argue that the substance has been selected by evolution because it possesses that type of biological activity (Firn, 200(D). However the vast majority of small-molecule plant antimicrobials are agents with weak or narrow-spectrum activities, which has led to suggest that "antimicrobials" actually have other functions in the plant and their low-level activity is accidental and largely irrelevant. Others have pointed out that at present there is a lack of a solid rationale for making a functional assignment for the vast majority of plant compounds that have been classified as antimicrobials (Tegos etal., 2002). Microorganims can become resistant to antibiotics and chemotherapeutic agents by various mechanisms. One of these mechanisms is by preventing the access of these agents to their target. The active pumping out (efflux) of drugs has been suggested as a major mechanism of tetracycline resistance in bacteria (Nikaido, 1998). It has been proposed that efflux mechanisms by multi-drug resistance i (MDR) provides a reasonable explanation for the apparent ineffectiveness of many antimicrobials in vitro (Tegos et al., 2002) It is essential to mention the importance of the role of light in mediating natural products' antibiotic activities. Many compounds, when excited by light, 13 display toxicity towards living cells or organisms. Light mediated biological activities of natural products from plants and fungi have been reviewed by Towers and co-workers (1997). For instance, thiarubrine A, a known constituent of a number of species in the Asteraceae, displays higher activity when exposed to light against C. albicans (Towers et al., 1985). Furthermore thiarubrines showed antiviral activity only against membrane containing viruses suggesting that membrane components were the biological targets (Towers et al., 1997). 1.5. Microbial Diseases of the Skin The skin supports the growth of certain microbes, which are established as part of the normal microbiota. On superficial skin surfaces, certain aerobic bacteria produce fatty acids from sebum. These acids inhibit many microbes and allow better-adapted bacteria to flourish. Rashes and lesions on the skin do not necessarily indicate an infection of the skin; in fact, many systemic diseases affecting internal organs are manifested in skin lesions (Tortora, 2002). The normal flora of the skin consists primarily of gram-positive bacteria restricted to a few groups. The lack of colonisation of gram-negative bacteria on the skin is probably due to their inability to compete with gram-positive organisms that are better adapted to the dry conditions of the skin. 1.5.1. Bacteria Staphylococcus and Streptococcus are amongst the most frequent causes of bacterial skin diseases. The genus Staphylococcus contains common 14 pathogens of humans and animals. S. aureus a yellow-pigmented form is associated with pathological conditions, including boils, pimples, and impetigo (superficial skin infection) and folliculitis (infection of hair follicle). Because in the hospital environment, patients, hospital staff members, and visitors carry S. aureus, the danger of infection is important. It is the most common cause of surgical wound infections and pneumonia, and the second most common cause of blood infections. Many strains are highly virulent and are also resistant to common antibiotics. Extensive use of antibiotics has resulted in the natural selection of resistant strains of Staphylococcus aureus such as methicillin resistant strains (Madigan, 2000). Like other staphylococci Streptococcus pyogenes can cause the local infection impetigo. When it infects the dermal layer of the skin, it causes a serious disease, erysipelas, that can eventually lead to local tissue destruction and even enter the bloodstream, causing septicemia. Pseudomonas aeruginosa can also cause dermatitis (superficial rash) and otitis externa, however, infection by P. aeruginosa is rare in healthy people. These aerobic gram-negative rods are widespread in soil and water and are considered a model of an opportunistic pathogen (Tortora, 2002). 1.5.2. Yeast. Candida albicans. Over the past few years there has been an increase in the incidence of fungal infections in several countries. In the commensal state, Candida spp. live as benign members of the microflora of healthy individuals. A s a commensal, C. 15 albicans causes no disease. Candida spp. are "carried" in the oral cavity, gastrointestinal tract, skin, anus and groin of healthy individuals, and also in the vaginal canal and vulva of healthy women (Soli, 2002). However, this yeast can also be considered an opportunistic pathogen. In situations where there is a decrease in host defences, it is able to access different locations in the human body and cause candidosis (Navarro-Garcia et al., 2001). Candida infections of virtually every tissue in the human body have been reported, although by far the most common manifestations of candidosis are superficial lesions, especially infections of the mucous surfaces of the mouth and vagina, and in moist areas of skin. Of the dimorphic fungi, C. albicans has the highest incidence of disease. It is a common cause of nosocomial infection; 70% of all women experience Candida vaginitis at least once in their lives and 70% of AIDS patients manifest oropharyngeal candidosis (Rooney and Klein, 2002). Candida has a unicellular mode of development and the genus comprises more than 150 species, whose main common feature is the absence of any sexual form (Odds, 1988). One of the main characteristics of this pathogenic yeast is its ability to switch from a unicellular to a hyphal mode of growth, a property called dimorphism. Dimorphism is triggered in response to changes in certain i environmental conditions, such as temperature, pH or serum availability. It is thought that it is this change which allows the organism to invade tissues and, therefore contributes to the virulence of this micro-organism (Navarro- Garcia et al., 2001). 16 A s t h e ho s t b e c o m e s i m m u n o c o m p r o m i s e d o r the c o m p e t i n g f l o r a a r e a l t e r ed , th i s oppo r t un i s t b e g i n s d i s p l a y i n g c e l l s of t he h y p h a l m o r p h o t y p e ( R o o n e y a n d K l e i n , 2 0 0 2 ) . D i m o r p h i s m in C. albicans h a s c a u s e d e n o r m o u s in te res t b e c a u s e of t he p r e s u m p t i o n that h y p h a f o r m a t i o n is a p r o c e s s of p a t h o l o g i c a l s i g n i f i c a n c e . O n e r a t i ona l e is that t he h y p h a l f o r m u n i q u e l y e x p r e s s e s m o l e c u l a r v i r u l e n c e f a c t o r s , w h i c h a s s i s t in t h e p a t h o g e n e s i s of c a n d i d o s i s . It i s not ce r t a i n w h e t h e r y e a s t s o r h y p h a e c o n s i s t e n t l y p l a y a s u p e r i o r ro le in the p a t h o g e n e s i s o f c a n d i d o s i s . B o t h m o r p h o l o g i c a l f o r m s o f t h e f u n g u s s e e m to h a v e t he ab i l i ty to in it iate a n d to s u s t a i n p a t h o l o g i c a l r e s p o n s e s in m a m m a l i a n ho s t s , but it is l i ke ly that o n e f o r m m a y b e be t te r a d a p t e d t h a n t he o t h e r to s u r v i v e in s p e c i f i c e c o l o g i c a l m i c r o n i c h e s in vivo. H o w e v e r , t he g r o w t h of C. albicans in h y p h a l f o r m is not i n va r i ab l y a s s o c i a t e d w i th t i s s u e i n v a s i o n in vivo, no r is its g r owth in t he y e a s t f o r m i nva r i ab l y a s s o c i a t e d w i th c o m m e n s a l s ta tu s . It is b e l i e v e d that a l l f o r m s a r e r equ i r ed to m a i n t a i n a n i n fec t i on . T h e r e is a g r o w i n g b o d y o f e v i d e n c e l i nk ing p h e n o t y p i c s w i t c h i n g w i th p a t h o g e n i c i t y o f th i s f ung i ( R o o n e y a n d K l e i n , 2 0 0 2 ) . F o r s o m e a u t h o r s th i s d i m o r p h i s m c o n s t i t u t e s a v i r u l e n c e trait per se, but is a l s o c o - r e g u l a t e d w i th o t h e r v i r u l e n c e f a c t o r s , w h i c h a r e a s s o c i a t e d w i th c e l l u l a r m o r p h o l o g y (E rns t , 2 0 0 0 ) . T h e t h r ee m a j o r g r o u p s o f an t i f unga l a g e n t s in c l i n i c a l u s e , a z o l e s , p o l y e n e s , a n d a l l y l a m i n e / t h i o c a r b a m a t e s , a l l o w e the i r a n t i f u n g a l ac t i v i t i e s to inh ib i t ion of s y n t h e s i s of o r d i r ec t i n te rac t i on w i th e r g o s t e r o l . E r g o s t e r o l is t he p r e d o m i n a n t i s o p r e n o i d l ip id c o m p o n e n t of the f u n g a l c e l l m e m b r a n e . C u r r e n t a v a i l a b l e t h e r a p i e s re ly m a i n l y o n t h e p o l y e n e a n d a z o l e ( s yn the t i c ) c o m p o u n d s . 17 The polyene amphotericin B is the most effective antifungal drug available, but its narrow therapeutic index continues to limit its clinical utility (Ghannoum and Rice, 1999). Some concepts like antibiotic resistance bacteria leading to hyper-virulent strains have been incorporated into interpretations of fungal pathogenesis. However epidemiological data have not revealed emergence of stable drug-resistant or hyper-virulent strains. Phenotypic switching in response to environmental cues suggests the need to identify the built-in mechanisms these fungi employ for rapidly adapting to challenges (Soli, 2002). 1.5.3. Aspartic Proteases as Virulence Factors It is very difficult to define virulence for a commensal organism like C. albicans. The concept that virulence is conferred by virulence factors applies best to pathogens that are free-living and able to cause disease in hosts with intact immunity (Casadevall and Pirofski, 2001). This underscores the difficulty in applying classical virulence factors concepts to microbes like C. albicans whose pathogenicity is limited mostly to immunocompromised hosts. Nonetheless it is possible to point out several factors that seem to contribute to the pathogenicity of C. albicans. Not only factors depending on the micro-organism are involved but also others that are strongly dependent on the host, such as tissue-specific differences in infection susceptibility (Navarro-Garcia et al., 2001). With this note of caution in mind it is important to mention the prominence of secreted aspartic proteases (SAPs) amongst the putative 18 virulence factors. S A P s constitute a family of isoenzymes encoded by at least nine genes (SAP) that are diferentially expressed in vitro and in vivo (De Bernardis et al., 1999). C. albicans strains with disruption of secretory aspartic proteases [SAP 1 to SAP 6) were assessed in a rat vaginitis model. Null sap 1 to sap 3 but not sap 4 to sap 6 mutants lost most of the virulence of their parental strain (De Bernardis etal., 1999). Inhibition of S A P s with the aspartic protease inhibitor pepstatin A prevented the initial penetration of C. albicans through mucosal surfaces but not the dissemination of the fungus once the cells had reached the blood vessels (Fallon et al., 1997). Moreover, pepstatin A prevented C. albicans invading and causing tissue damage in oral, vaginal and skin experimental infection models (De Bernardis et al., 1995). HIV aspartic protease inhibitors can inhibit C. albicans adherence to epithelial cells (Borg-von Zepelin et al., 1999). Extracellular matrix and host surface proteins (keratin, collagen, fibronectin) are efficiently degraded by S A P 2. Defence host proteins such as salivary lactoferrin and almost all immunoglobulins, including secretory IgA, which is normally resistant to most bacterial proteases, can also be hydrolysed by S A P 2 (Hube, etal., 2001) In a mouse gastrointestinal tract model, no experimental support was found for a unique role of individual S A P s . It would seem that S A P s action in colonisation is collective and that absence of one S A P is compensated for by others (Kretschmar et al., 2002). Secreted aspartic proteases by Candida 19 represent a potential target for drug intervention of the disease, and inhibition of S A P s has been proposed as a new approach on the treatment of candidosis (Zhang et al., 2002). Natural products like xanthones and benzophenones present in the Guttiferae family have been reported to have effects against C. albicans secreted aspartic proteases (Zhang et al., 2002) but no powerful plant-derived inhibitor has been commercially developed. 1.5.4. Viruses In regards to viral disease, herpes simplex virus type 1 is the causative agent of herpes simplex. Frequently, this infection is subclinical, but in many cases causes gingivostoma ("fever blister" or "cold sore") - vesicles around the mouth (Tortora, 2002). Herpes simplex virus (HSV) most frequently produces recurrent painful vesicular eruptions of the skin and mucous membranes. Both HSV-1 and HSV-2 can cause severe protracted and disseminated disease in immunocompromised persons as well as necrosis of infected cells, which is accompanied by a vigorous inflammatory response. Formation of clusters of painful ulceration vesicular lesions on the skin or mucous membranes are the most frequent manifestation of HSV infection (Rubin, 1998). Human poliovirus is a single stranded non-enveloped RNA virus in the family Picornaviridae. This virus will be used to test the different plant extracts because of its resistance to chemical compounds and for comparative purposes since H S V is a DNA virus. 20 1.6. The Burden of Antibiotic Resistance Because microbes have the genetic flexibility to develop resistance to any antimicrobial agent it is necessary to accept that the use of antimicrobial therapy is always going to be compromised over time. The impact of antibiotics in Western society is quite important and nowadays, it is a matter of great concern that no new chemical classes of active antibiotics have been successfully introduced into the clinic for over 30 years (Hancock and Knowles, 1998). Although there are many instances of spontaneous mutation in the development of resistance to antibiotics (e.g. streptomycin-resistant Mycobacterium tuberculosis), mutation is more the exception than the rule. This is because the genetic basis of most antibiotic resistance among clinically significant bacteria is due to the dual process of gene acquisition and gene horizontal transfer (Mazel and Davies, 1999). In the majority of cases, acquisition from exogenous (and still largely unidentified) sources was the primary mechanism by which bacteria obtained genes encoding resistance to antibiotics (Davies, 1994). The increasing threat of drug-resistant pathogens is causing a renewed interest in the discovery of novel antibiotics. A current trend is to identify essential genes/proteins that are not targets for known antibiotics, and use the purified proteins to search for potent binding ligands from natural sources or combinatorial libraries. A general problem encountered with this approach is the 21 presence of numerous multidrug pumps found in all bacteria and yeast studied so far (Hsieh et al., 1998). Preventing the access of drugs to the target is one of the various mechanisms microorganisms possess to become resistant to antibiotics. Ubiquitous multidrug resistance (MDRs) pumps, membrane translocases that extrude structurally unrelated toxins from the cell, confer a general and effective defence against antimicrobials' (see Figure 1.1) (Nikaido, 1998). Amphiphilic drug transporter i Figure 1.1. Gram-positive Bacterial Multidrug Pump: In Gram-positive bacteria, drugs come in unhindered, and are pumped out into the medium (dashed arrows). From Nikaido (1998). 22 This mechanism involves the active pumping out (efflux) of drugs, a process that has been known since the discovery by Levy and his associates (1992) of active efflux as a major mechanism of tetracycline resistance in bacteria. In recent years, it was found that some of the efflux systems pump out more than one substrate. Nor A is a multidrug pump that belongs to the major facilitator super-family (MFS) of proteins, which use proton motive force as the driving force for efflux (Nikaido, 1998). The NorA MDR pump of S. aureus protects the cells from norfloxacin and a number of amphipathic cations, such as the common disinfectants benzalkonium chloride and cetrimide (Hsieh et al., 1998). A mutant of S. aureus with a knockout in the norA gene coding for the MDR (multidrug resistance) pump has a substantially increased sensitivity to a large number of antimicrobials, including therapeutically significant compounds (Hsieh etal., 1998). It has been argued that multidrug resistance pumps evolved in response to natural antimicrobial amphipathic cations. For this reason it has been suggested that cells with mutations abolishing the function of their multidrug resistance pumps could be used to increase the sensitivity of screens for new antimicrobial agents (Lewis, 1999). Berberine alkaloids, which are widely spread in the plant world and are prominent in the Ranunculaceae family, appeared to be good substrates for the NorA multidrug resistance pump and for the QacA multidrug resistance pump of S. aureus. Consistent with this, mutations knocking out multidrug resistance pumps were found to turn berberine into a very strong 23 antibiotic. Lewis and co-workers have shown a case (probably the first) of molecular synergy in the medicinal plant Berberis fremontii (Berberidaceae) used in Native North-American traditional medicine. This plant produces the weak antibacterial berberine (Figure 1.1) and a potent multidrug resistance pump (MDR) inhibitor identified as 5' - methoxyhydnocarpin (5' -MHC)(Figure 1.2). Berberine used on its own is rendered ineffective due to the presence of the NorA MDR pump. However, when used in conjunction with 5' - M H C , berberine exhibits good activity (MIC=1 pg/mL) (Stermitz etal., 2000). ^ W / ° C H 3 \ ^ O C H 3 Figure 1.2. Structure of Berberine 24 Figure 1.3. Structure of 5'-methoxyhydnocarpin D (5' -MHC) Their experiments showed how two different components of a medicinal plant act in synergy, with one compound disabling a resistance mechanism and potentiating the antimicrobial activity of the antibiotic substance. Having found natural synergy between berberine and a MDR inhibitor, Tegos and co-workers (2002) tested the general possibility that plant antimicrobials are potentially effective if they are delivered into the pathogen cell while inhibiting the efflux mechanism. A marked increase in the levels of activity of plants antimicrobials was observed, supporting the hypothesis for the apparent ineffectiveness of many antimicrobials in vitro. This is a fascinating illustration that might explain why chemical extracts sometimes present better activity than isolated compounds. 25 1.7. Culture and Environment Colombia is a country with high cultural and geographical diversity. A census done in 1997 by the National Department of Statistics (DANE) shows a population of 708,860 indigenous people, equivalent to 1,75% of the Colombian population. These populations consist of 80 different ethnic groups, who have territorial rights over 24,5%(279,487 square kilometres) of the national territory (Arango and Sanchez, 1998). 1.7.1. Huitoto Huitoto people live in the Amazon basin that constitutes a large territory that covers 35% of the Colombian land surface and the 61% of the natural forests. Presently this region is inhabited by 44 different ethnic groups and it is considered the most ethnically diverse in the country (Arango and Sanchez, 1998). The Huitoto people are also known as murui or muinane. They constitute a typical tribal group of the Amazonian tropical rainforest. They make their living from horticulture, hunting, fishing and fruit collecting and their main transportation means are by foot or canoe. The main crops are yuca (Manihot esculenta), pineapple (Ananas comosus), plantain (Musa paradisiaca), coca (Erythroxylum coca), tobacco (Nicotiana tabacum), corn (lea mays), sugarcane (Saccharum officinarum) and endemic trees. Each family has two swidden plots: one in production and the other in a growing stage. Both are located within the tropical 26 rainforest, are distant from one another and several hours away from the main hamlet. The Huitoto people believe that ailments are caused by supernatural powers. To cure them, they bum aromatic plants around the sick person and use their mouth to extract foreign bodies responsible for the disease. In the early part of the twentieth century Huitoto were mercilessly exploited during the rubber boom, by the Peruvian Casa Arana -Peruvian Amazon Rubber Company. Violence caused the death of approximately 40,000 Indians (Llanos and Pineda, 1982). Yet the remnants of these once very populous tribes kept their language, culture and knowledge about the properties of plants. Today the Huitotos are the second largest indigenous group in the Colombian Amazon (around 6,245 people) (Arango and Sanchez, 1998). 1.7.2. Kamsa The Kamsa and Inga indians occupy part of a small highland valley 2,200 m. above sea level in southern Colombia called the Sibundoy Valley. According to the last census, Kamsa people are approximately 4,022 in number in an area of 4,402 ha. (Arango and Sanchez, 1998). The climate of the valley is cool and exceptionally humid. A large non-Indian population also inhabits the valley, and i although each group tends to remain apart from the other there is a significant interaction between them, particularly in economic matters. These settlers dominate the local economy and has the best farmland. 27 The Kamsa and Inga people have become acculturated as a result of schooling, financial transactions and other activities with the white population. All but a few elders are fluent in Spanish, yet they continue to maintain separate identities probably due to the use of traditional language, traditional clothing and separate residences. Most Kamsa and Inga people have knowledge of herbal remedies and their gardens include several herbs for the treatment of common and minor illnesses. The shamans have received formal training in the use of herbal remedies and the ingestion of the hallucinogen yage (Bannisteriopsis caapi) is of major importance in diagnostic and therapeutic procedures for illnesses believed to have supernatural causes (Chaves et al., 1995) Apart from the above, the Kamsa and Inga people have several western medical and para-medical services available in the event of illness yet the healer continues to be regarded more highly than physicians provided by the Government. 1.7.3. Sikuani The tropical rainforest of Mataven is located north of the Colombian Amazon and surrounded by the Vichada, Orinoco, Guaviare and Chupave Rivers area which is known as the Orinoco region. The Sikuani are approximately 20,544 people inhabiting an area of 2,117,532 ha (Arango and Sanchez, 1998). They are semi nomads and their culture is strongly linked to ecological factors. During dry season the Sikuanis are highly nomadic and rely on hunting, fishing 28 and fruit collection while in the rainy season they become sedentary and rely upon swidden plots previously established in the higher areas of the basin (Chaves et al., 1995). As in many other cultures, the shaman cures ailments, provides counseling and teaches prayers that bring good luck, good weather or the opposite. The knowledge associated with the traditional use of medicinal herbs by the Sikuanis is not widespread when compared to other indigenous groups in Colombia ( Chaves et al., 1995). Very few ethno-botanical studies have been carried out in the region. 1.7.4. Afro-Colombian Communities The Pacific littoral is one of the world's rainiest areas, annual precipitation varying between 5,000 and 12, 000 mm/year although dry and rainy seasons occur. It is a long (1,300 km) and wide corridor and corresponds to 7% of the national territory (Arango and Sanchez, 1998). The temperature is high with humidity near saturation all of the time. Afro-Colombian communities inhabit the area since the 16 t h century and have shared the environment and knowledge with the natives since then. They f have profited from plants to treat ailments, obtain food and construction materials since the beginning of the slave trade. Like other communities of African origin inhabiting tropical regions of the world, they share extremely poor living standards despite residing in extremely rich natural and cultural environments. 29 Until today few ethnobotanical studies have taken place in the region (Caballero, 1995). As in many communities of African descent, certain features characterize most of their healing traditions: these include theories related to the spiritual realm, the capacity to identify symptoms associated with specific diseases and the ability to prescribe culturally acceptable treatments (Voeks 1993). Cures are most often effected through votive offerings to the ancestors and spirits, observance of taboos, fasting and seclusion, and prescriptions of medicinal plants (Voeks 1993). Lumber operations to secure the availability of large softwoods are quite important to the cash economy of the region and the country. Timber from the interior must be floated down the main rivers to the mangrove delta. Towns emerging along these riverbanks rise and fall in size and importance according to the vicissitudes of external world markets. People inhabiting the wet Pacific littoral have adjusted their lives to such boom-bust economies (Whitten, 1974). 30 1.8. References Arango, R. and Sanchez, E. 1998. Los pueblos indigenas de Colombia: Desarrollo y territorio. Tercer Mundo Editores. Santafe de Bogota, p. 19. Balick, M. and Cox, P. A. 1996. Plants, people and culture: the science of ethnobotany. 1 s t Edition. New York Scientific American Library. New York. p.3. Borg-von Zepelin, M., Meyer, I., Thomssen, R., Wurzner, R., Sanglard, D., Telenti, A. Monod, M. 1999. HIV-Protease Inhibitors reduce cell adherence of Candida albicans strains by inhibition of yeast secreted aspartic proteases. The dournal of Investigative Dermatology 113 .747-751. Caballero, R. 1995. La etnobotanica en las comunidades negras e indigenas del delta del rio Patia. 1 s t. Edition. Abya-Yala Editores. Quito, pp.15-17. Casadevall, A . and Pirofski, L. 2001. Host-Pathogen interactions: the attributes of virulence. The dournal of Infectious Diseases 184 : 337-344. i Chaves, A, Morales, J , and Calle, H. 1995. Los indios de Colombia. 1 s t Edition. Editorial M A P H R E . Madrid, pp.283-314. 31 Cowan, M. M. 1999. Plant products as antimicrobial agents. Clinical Microbiology Reviews 12: 564-582. Davies, J . 1990. What are antibiotics? Archaic functions for modern activities. Molecular Microbiology 4:1227-1232. \ Davies, J . 1994. Inactivation of antibiotics and the dissemination of resistance genes. Science 264: 375-382. Davis, W. 1996. One River. Explorations and Discoveries in the Amazon Rain Forest. Simon and Schuster. New York. p. 535. De Bernardis, F., Cassone, A., Sturtevant, J . and Calderone, R. 1995. Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis. Infection and Immunity 63 :1887-1892. De Bernardis, F. Arancia, S., Morelli, L. Hube, B., Sanglard, D., Schafer, W. and Cassone, A. 1999. Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP2, are virulence factors for Candida vaginitis. The i dournal of Infectious Diseases 179: 201-208. Ernst, J .F. 2000. Transcription factors in Candida albicans - environmental control of morphogenesis. Microbiology 146 : 1763-1774. 32 Fallon, K., Bausch, K., Noonan, J . , Huguenel, E. and Tamburini, P. 1997. Role of aspartic proteases in disseminated Candida albicans infection in mice. Infection and Immunity 65:551-556. Farnsworth, N. R., Akerele, O., Audrey, S., Soejarto, D.D. and Guo, Z. 1985. Medicinal Plants in Therapy. Bulletin of the World Health Organization 63:965-1170. Farnsworth, N. R. 1990. The role of ethnopharmacology in drug development: In: Bioactive compounds from plants. Wiley. Chichester, pp. 2-21. Firn, R.D. and Jones, C G . 2000. The evolution of secondary metabolism: A unifying model. Molecular Microbiology 37: 989- 994. Furst, P. T. 1995. "This little book of herbs": Psychoactive plants as therapeutic agents in the Badianus Manuscript of 1552. In: Ethnobotany: evolution of a discipline. Edited by: Schultes, R. E. and Reis Siri von. Dioscorides Press. Portland.pp.108-130. Ghannoum, M. A. and Rice, L. B. 1999. Antifungal Agents: Mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clinical Microbiology Reviews 12: 501-517. 33 Grifo, F., Newman, D. Fairfield, A. S., Bhattacharya, B. and Grupenhoff, J . T. 1997. The Origins of Prescription Drugs. In: Biodiversity and Human Health. Edited by: Grifo, F. and Rosenthal, J . Island Press Washington D.C. pp.131-163. Hancock, R and Knowles, D. 1998. Are we approaching the end of the antibiotic era? Current Opinion in Microbiology 1: 493-494. Hsieh, P-C. Siegel, S. A. Rogers, B. Davis, D. and Lewis, K. 1998. Bacteria lacking a multidrug pump: A sensitive tool for drug discovery. Proceedings of the National Academy of Sciences USA. 95: 6602-6606. Holmsted and Bruhn. 1995. Purpose of Ethnopharmacology. In: Ethnobotany:Evolution of a Discipline. Edited by: Schultes.R.E. and Von Reis , S. Portland. Dioscorides Press, pp.338-342. Hube, B. and Naglik, J . 2001. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147: 1997-2005. Hudson, J . B. 1990. Antiviral compounds from plants. C R C Press. Boca Raton. i p.200. 34 Janzen, D.H. 1978. Complication in interpreting the chemical defences of trees against tropical arboreal plant eating vertebrates. In: The ecology of arboreal foltvores-eating vertebrates. Edited by: Institutional Press. Washington, D.C. pp. 73-84. Johns, T. 1990. With bitter herbs they shall eat it. Edited by: The University of Arizona Press. Tucson, pp. 1-3. Kretschmar, M., Felk, A., Staib, P. Schaller, M., HeG, D., Callapina, M., Morschhauser, J . , Schafer, W., Korting, H.C., Hof, H., Hube, B. and Nichterlein, T. 2002. Individual acid aspartic proteinases (Saps) 1-6 of Candida albicans are not essential for invasion and colonization of the gastrointestinal tract in mice. Microbial Pathogenesis 32: 61-70. Levy, S.B. 1992. Active efflux mechanisms for antimicrobial resistance. Antimicrobial Agents and Chemotherapy 36:695-703. Lewis, K. 1999. Multidrug resistance: Versatile drug sensors of bacterial cells. Current Biology 9: R403-R407. Llanos, H. and Pineda, R. 1982. Etnohistoria del Gran Caqueta, Siglos XVI-XIX. Edited by: Fundacion de Investigaciones Arqueologicas. Banco de la Republica. Bogota, pp. 47-55. 35 Madigan, M. T. 2000. Epidemiology and Public Health Microbiology. Chp. 22. In: Brock biology of microorganisms. 9 t h Edition. Prenticel Hall. New Jersey.pp. 945-777. Mann, J . 2000. Murder, magic and medicine. 2 n d Edition. Oxford University Press. New York. p. 256. ^ Mazel, D. and Davies, J . 1999. Antibiotic resistance in microbes. Cellular and Molecular Life Sciences 56: 742-754. Moerman, D.E. 1998. Native American Ethnobotany. Timber Press. Portland, p. 927. Navarro-Garcia, F., Sanchez, M., Nombela, C. and Pla, J . , 2001. Virulence genes in the pathogenic yeast Candida albicans. FEMS Microbiology Reviews 25: 245-268. Nikaido, H. 1998. Multiple antibiotic resistance and efflux. Current Opinion in Microbiology 1: 516-523. Newman, D.J., Cragg, G. M. and Sander, K. M. 2000. The influence of natural products upon drug discovery. Natural Products Reports 17: 215-234. 36 Odds, F. C. 1988. Biological Aspects of Yeast Pathogens. In: Candida and Candidosis. 2 n d . Edition. Bailliere Tindall. London, p.7. Rooney, P.J . and Klein, B.S. 2002. Linking fungal morphogenesis with virulence. Cellular Microbiology 4: 127-137. Rubin, E. 1998. Pathology 3 r d Edition. Lippincott- Raven Publishers. Philadelphia. pp.368-370. Sandberg, F. and Corrigan, D. 2001. Natural Remedies. Their origins and uses. Taylor and Francis. London, p. 39. Simpson, B. B. 2001. Medicinal Plants. Chp 11. In: Economic Botany: Plants in our World. 3 r d Edition. McGraw-Hill. New York. pp. 263-285. Soli, D.R. 2002. Candida commensalism and virulence: the evolution of phenotypic plasticity. Acta Tropica 81: 101-110. Stermitz, F. R., Lorenz, P., Tawara, J . N., Zenewicz, L.A. and Lewis, K. 2000. Synergy in a medicinal plant: Antimicrobial action of berberine potentiated by 5' -methoxyhydnocarpin, a multidrug pump inhibitor. Proceedings of the National Academy of Sciences 97: 1433-1437. 37 Tang, W. and Eisenbrand, G. 1992. Chinese Drugs of Plant Origin. Chemistry, Pharmacology, and Use in Traditional and Modern Medicine. Springer-Verlag. Berlin, p 1. Tegos, G. , Stermitz, F. R., Lomovskaya, 0 . and Lewis, K. 2002. Multidrug pump inhibitors uncover remarkable activity of plant antimicrobials. Antimicrobial Agents and Chemotherapy 46: 3133-3141. Tortora, G. 2002. Microbial diseases of the skin and eyes. Chp 21. In: Microbiology: an introduction. 7 t h Edition. Benjamin Cummings. San Francisco. pp.578-571. Towers, G . H. N., Abramowski, Z., Finlayson, A. J . and Zucconi, A. 1985. Antibiotic properties of thiarubrine A, a naturally occurring dithiacyclohexadiene polyine. Planta Medica 3: 185-290. Towers, G.H.N. , Page, J .E . and Hudson, J . B. 1997. Light-mediated biological activities of natural products from plants and fungi. Current Organic Chemistry 1: 395-414. 38 Unschuld, P. U. 1988. Culture and pharmaceutics: some epistemological observations on pharmacological systems in ancient Europe and Medieval China. In: The context of medicines in developing countries. Studies in pharmaceutical anthropology. Edited by: Sjaak Van Der Geest and Susan Reynolds Whyte. Kluwer Academic Publishers. Dordrecht, p. 188. \ Voeks, R. 1993. African medicine and magic in the Americas. Geographical Review 8 3 : 66-78. Whitten Jr, N. E. 1974. Black Frontiersmen. A South American case. John Wiley and Sons. New York. p. 32. Zhang, Z. ElSohly, H. N. Jacob, M., Pasco, D. S., Walker, L. A. Clark, A. M. 2002. Natural Products inhibiting Candida albicans secreted aspartic proteases from Tovomita krukovii. Planta Medica 68: 49-54. 39 Chapter 2 Field Collection of Colombian Medicinal Plants "People with pimples and ulcers on the skin, have spoiled blood. It is necessary to clean and purify this blood so they can be cured..." \ Domingo Chasoy Popayan, Colombia 2.1. Introduction Nothing is further from reality than the idyllic image of the ethnobotanist immersed deep in the forest in search of the shaman who will reveal his/her herbal medicinal secrets to the outsider. This image probably has its origins in earlier accounts from botanists exploring the north-western Amazon. The name of Richard Evans Schultes is the one that comes first to my mind in regard to the search of herbal remedies based on indigenous traditional knowledge. His book entitled "The Healing Forest" (Schultes and Raffauf, 1990) is an impressive treatise of medicinal plants used for medicinal purposes by Huitoto, Muinane and Bora people to name just a few. Schultes, in his introduction, stated the importance of recording this knowledge before it disappeared but assumed that traditional knowledge was not and is not subject to change and consequently can be recorded once and for all. In his opinion the body of knowledge about therapeutical practices in which plant uses are entwined was static. In other words, the indigenous medical 40 s y s t e m s w e r e ou t s i de the n o r m a l p r o c e s s e s of cu l tu ra l t r a n s f o r m a t i o n . T h e n a m e s of d i f fe rent t r i be s w e r e t h e r e b e c a u s e of t a x o n o m i c c o m p u l s i o n a n d m e d i c a l p r a c t i c e s h a d n o c o n n e c t i o n w h a t s o e v e r to the i r m y t h o l o g y o r re l i g i on w h i c h m i gh t h a v e e x p l a i n e d the u s e o f a p lant. In shor t , h e s a w i n d i g e n o u s p o p u l a t i o n s a s p a s s i v e e l e m e n t s in a n idy l l i c i n te rp l ay w i th n a t u r e a n d r e g a r d e d t h e m a s non -h i s t o r i c a l i nd i v i dua l s . I nd i genou s c o m m u n i t i e s w e r e t h e n t r a n s f o r m e d into s ta t i c ent i t i e s w h o s e k n o w l e d g e w a s s u s c e p t i b l e to b e r e g i s t e r ed . O p p o s i n g th i s v i e w , I c o n s i d e r that it is u r gen t to d i s c u s s t he u s e of m e d i c i n a l p l an t s in a n h i s to r i ca l c on tex t . I ndeed w h e n o n e c o n s i d e r s the p e r s e c u t i o n , d i s p l a c e m e n t a n d ann i h i l a t i on of t he i n d i g e n o u s p e o p l e a l o n g the C a q u e t a R i v e r d u r i n g t h e r u b b e r e xp l o i t a t i on , o n e m u s t w o n d e r a b o u t t h e c o n c e p t o f ident i ty a n d h o w th i s c o n c e p t i s r e f l e c t e d in t he i r t r ad i t i ona l m e d i c a l s y s t e m s . T h e i n f l u e n c e of t he C a t h o l i c C h u r c h in r e g i o n s l i ke t he S i b u n d o y va l l e y led to a cu l tu ra l f u s i on that d i rec t l y i n f l u e n c e d the m e d i c a l s y s t e m s of the K a m s a a n d Inga p e o p l e . T h e s e h i s to r i ca l c i r c u m s t a n c e s s e e m to h a v e b e e n c o m p l e t e l y d i s r e g a r d e d bo th by b o t a n i s t s a n d e t h n o b o t a n i s t s . T h e q u e s t i o n o f h o w h i s to r i ca l p r o c e s s e s h a v e t r a n s f o r m e d t rad i t i ona l m e d i c a l s y s t e m s h a s not b e e n a d d r e s s e d . It is impo r t an t to n o t e that t he p r e s e n t c h a p t e r d o e s no t i n c l u d e a h i s t o r i ca l b a c k g r o u n d o f t h e i nd i v i dua l s w h o h e l p e d m e d u r i n g t he f i e l d c o l l e c t i o n . T h u s I d o not c o n s i d e r th i s f i e ld c o l l e c t i o n to b e a n e t h n o b o t a n i c a l o n e but r a the r a c o l l e c t i o n of p l an t s t rad i t i ona l l y u s e d in the t r e a t m e n t of m o r b i d s k i n c o n d i t i o n s 41 but devoid of a cultural context that would explain their uses. I consider this a drawback whose discussion will be included in chapter six. I followed the ethnobotanical practice of interviewing, collecting and recording plant uses. The research reported in this chapter may have a limited value from the anthropological point of view, but it gave me the opportunity to compare plant uses in (different cultural and geographical communities in Colombia. 2.2. Materials and Methods 2.2.1. Plant Collection The plants were collected at one site each in four different areas in Colombia (Fig 2.1) guided by local healers and villagers. Information about the therapeutic properties of the plants was gathered by participating in domestic activities and interviewing traditional healers (curanderos) and knowledgeable villagers about those plants used to treat cutaneous conditions of any kind. The information gathered included native and/or common names, part(s) used and recipes. Plant material was air-dried in the shade and a set of labelled voucher herbarium specimens was made for each collection; these vouchers were filed in i the National Herbarium of Colombia (COL) and the Sinchi Institute Herbarium (Santafe de Bogota, Colombia). 42 2.2.2. Col lect ion Sites Distribution of the collection sites were as follows 44 BAJO CALIMA REGION (Valle Department) Afro-Colombian community This area lies in the Choco biogeographic region south of the Calima River, at 03 057'-04 0-10' N and 77°0T-12'W on the Pacific Coast of Colombia near the port of Buenaventura, in the Valle Department. It occupies approximately 60,000 ha, at an elevation between 0 and 300 m and receives 7,000-8,000 mm of rain annually (Motta, 1997). The Bajo Calima site has been inhabited by Afro-Colombian communities for many generations and only few years ago it became a timber concession (1950-1995) to the company Carton de Colombia. The plant collection was carried out along the Calima River near the coast. Hubert Murillo and his father, Adriano Murillo, who lived in the region, guided the collection of the plants. MATAVEN FOREST (Vichada Department) Sikuani communiy The Sikuani reserves are mainly located in the Departments of Vichada and Meta. There are 20,544 people in an area of 2,117,532 ha. Mataven forest is north of the Colombian Amazon near Cumaribo in the Vichada Department. The area has an elevation of 166 m and precipitation ranging from 1,500 to 2,000 mm annually (IGAC, 1996). The plant collection carried out in the Cumaribo was guided by Elvira Carivan, a well-respected inhabitant of the region. 45 SIBUNDOY VALLEY (Putumayo Department) Kamsa and Inga communities The valley of Sibundoy lies near the eastern slope of the Andes at an elevation of 2,000 m. The climate of the valley is cool, very humid and with relatively little sunshine. The average annual rainfall is close to 2,136 mm (IGAC, 1996). Kamsa and Inga comunities in this valley inhabit an area of 4,402 ha. Although these communities live in the same territory, they belong to different linguistic families, the Kamsa and Quechua respectively. The plant collection was carried out in the village of Sibundoy and was guided by two traditional healers from the Kamsa community, Don Miguel Chindoy Mutumbajoy and Dona Clarita Buesaquillo. MIDDLE CAQUETA BASIN (Caqueta Department) Huitoto community The middle Caqueta basin is located in the north-western Colombian Amazon and covers 1, 300,000 ha, and is dissected by the Caqueta River, which is a tributary of the Amazonas and is one of the basin's main communication routes. The elevation ranges between 200 and 300 m and the region has an average annual rainfall of 3,000 mm (Galeano, 1992). Huitoto indigenous communities, along with Mirana, Muinane, and others, inhabit the middle region of the Caqueta River. Plant collection was carried out with two members of a Huitoto family, Oscar and Simon Roman. The collection site is located in Aguasal Creek, a small tributary of the Caqueta River. 46 2.3. Results. Field Ethnomedical Data A list of the collected plants is enumerated below, with voucher herbarium specimen number indicated in parentheses. A P I A C E A E Hydrocotyle umbellata L (Andres Lopez AL-57) Collection site: Sibundoy valley Common name: Chupana It is said that a mixture with animal fat will "suck" the infections out of the skin. A S T E R A C E A E Ambrosia artemisioides Willd. (AL-02) Collection site: Sibundoy valley Common name: Marco An infusion of leaves is prepared and used as a bath to "clean" the body. Conyza bonariensis (L.) Cronq. (AL-36) Collection site: Sibundoy valley i Kamsa name: Tacehsajch Annual herb in gardens, introduced, common. 47 It is used to treat dark spots on the face, acne and for the condition known as "carate" {Tinea alba). The plant is macerated and applied to the skin. Exposure to sunlight must be avoided. Tagetes erecta L. (AL-59) Collection site: Sibundoy valley Common name: Mapan A decoction of the flowers is used externally in the treatment of skin infections. Eupatorium glutinosum Lam. (AL-OT) Collection site: Sibundoy valley Common name: Matico An infusion of the leaves is prepared and used as a bath to "clean" the body. C E L A S T R A C E A E Goupia glabra Aubl. (Oscar Roman OR-73) Collection site: Middle Caqueta Basin Uitoto name: Jodina (Jodi= to get drunk; -na= tree) The extract of young leaves is used externally to treat blindness caused by cataracts and in the treatment of smallpox. It is used as a cicatricial when applied on the skin. 48 C L U S I A C E A E Vismia macrophylla K u n t h (AL - 05 ) C o l l e c t i o n s i te : M i d d l e C a q u e t a B a s i n C o m m o n n a m e : L a c r e U i to to n a m e : Y i i k o a i (Yii= that c h a n g e s co lo r ; -ai= s m a l l t ree ) T h e l ightly r o a s t e d l e a v e s a r e u s e d a s a d r e s s i n g o n s n a k e b i t e s a n d p l a c e d o n top o f t he w o u n d s . T h e ba r k i s u s e d e x t e r n a l l y in t h e t r e a tmen t o f c u t a n e o u s i n f e c t i on s w h i l e t he r e s i n i s u s e d f o r t he c o n d i t i o n k n o w n a s " c a r a t e " . T h e s a p is u s e d in the t r e a t m e n t of p r o b l e m s r e l a t ed w i th v i s i on . Symphonia globulifera L. f. ( A L - 88 ) C o l l e c t i o n s i te : B a j o C a l i m a C o m m o n n a m e : M a c h a r e A d e c o c t i o n o f t he ba r k is r u b b e d o n t h e s k i n f o r t r e a t m e n t o f c u t a n e o u s l e i s h m a n i a s i s , F A B A C E A E Senna reticulata (Wil ld.) H.S. Irwin et B a r n e b y ( A L - 3 3 ) C o l l e c t i o n s i te : B a j o C a l i m a i C o m m o n n a m e : G a l v e s A d e c o c t i o n o f the l e a v e s i s u s e d in the t r e a t m e n t of s k i n i n fec t i on s pa r t i cu la r l y in f u n g a l i n f e c t i on s that w h i t e n t he s k i n . 49 J U G L A N D A C E A E Juglans neotropica Diels (AL-89) Collection site: Sibundoy Valley Common name: Cascara de nogal An infusion of the bark is prepared and used as a bath to "clean" the body, as an antiseptic and to treat a condition known as "body rattening". A plant infusion is taken orally as a contraceptive. L E C Y T H I D A C E A E Eschweilera rufifolia S.A. Mori (OR-71) Collection site: Middle Caqueta Basin Common name: Carguero Uitoto name: Koduiro (kodui= that the bark is easily peeled off) The inner part of the bark is used externally as an anesthetic and as a cicatrizant in the treatment of wounds. It is also used in the preparation of vegetable salt. MALPIGHIACEAE Byrsonima verbascifolia (L.) DC. (EM-01) Collection site: Mataven Forest Sikuani name: Dorronae A bath is prepared with a mixture of the leaf and the bark of the root for treatment of cutaneous conditions. 50 MONIMIACEAE Siparuna guianensis Aubl. • (AL-87) Collection site: Mataven Forest Common name: Romadizo Sikuani name: Minisinae Cuiba name: Bibisine \^  The bark is macerated in water and used as a bath for skin diseases and headache. This maceration is also good to diminish fever. Also, an infusion from the leaves is used as a bath to "clean" the body. MYRISTICACEAE Iryanthera megistophylla A .C . Sm. (AL-29) Collection site: Bajo Calima Common name: Cabo de indio The decoction of the bark is used externally in the treatment of cutaneous leishmaniasis. Iryanthera tricornis Ducke (AL-12) Collection site: Middle Caqueta Basin i Common name: Cabo de hacha, mamita, sangretoro Uitoto name: Jtikai (Ji= to bend down; kat= refering to an elongated shape and smooth texture). The bark's aqueous extract is used internally in the treatment of measles. 51 Virola multinervia Ducke (AL-18) Collection site: Middle Caqueta Basin Common name: ambil de monte Uitoto name: Ukukat (uku= star; kai= refering to an elongated shape and smooth texture).The bark is used for the treatment of skin sores and fungal infections by crushing^ and boiling in water, and applying the decoction externally. M Y R T A C E A E Myrteola nummularia (Poir.) O. Berg (AL-86) Collection site: Sibundoy Valley Common name: Guayabilla Kamsa name: bchichaja Inga name: paramchichaja It is a well-known plant commonly sold in market places. A bath of the infusion of the whole plant is claimed to be useful in treatment of skin problems. P I P E R A C E A E i Piper lanceaefolium Kunth (AL-75) Collection site: Sibundoy Valley Common name: Cueche 52 The decoction of the leaves is taken as a bath to treat skin infections. Specifically, those that refer to a malignant cutaneous manifestation produced when one gets "the piss of a rainbow", a term which describes the drizzle that usually accompanies the presence of a rainbow. P O L Y G O N A C E A E Polygonum punctatum Elliott (AL-35) Collection site: Sibundoy Valley Common name: Picantillo Kamsa name: Fsteshaja Perennial herb in ditches, native, very abundant. A decoction of the plant is used externally in the treatment of skin infections. Because of its potency, it should be used with caution. As with Piper lanceaefolium, a bath of the infusion is taken for that purpose. According to Doha Clarita this preparation is used when parts of the body of a living individual are "rotten" . The Kamsa and Inga communities use the word "rotten" when they observe a change in color and swelling in the body tissue, but not necessarily to express the lost of form due to the action of bacteria, fungi etc. Don Miguel noted the benefits when the body is "watery". It is also used as an insecticide. 53 P T E R I D A C E A E Adiantum latifolium Lam. (AL-26) Collection site: Bajo Calima Common name: Montanero; Nena A decoction of the leaves is used to prevent tissue necrosis produced by the bite of the "bushmaster" snake. R H A M N A C E A E Ampelozizyphus amazonicus Ducke (AL-13) Collection site: Middle Caqueta Basin Muinane name: +mino-gaico Huitoto name: D+ineo; A+foio (+ is a high central vowel; it is pronounced by placing the tongue in the position of u and the lips in the position of /; Candre and Echeverri, 1996) The bark is scraped, added to water and vigorously shaken. The resulting foamy preparation is applied as an antiseptic on wounds. RUBIACEAE Duroia hirsuta (Poepp. & Endl.) K. Schum. (OR-74) Collection site: Middle Caqueta Basin It is used as a dye by mixing with other plants; the bark is used externally as a cicatrizant. 54 S I M A R O U B A C E A E Picrolemma sprucei Hooker f. (AL-03) Collection site: Middle Caqueta Basin Common name: Arbolito de casabe Uitoto name: Taingorai (taingo= cassava bread; -rai= wooden block) The leaf extract is applied over an area on the skin affected by an infection. S O L A N A C E A E Solanum sp. (AL-85) Collection site: Sibundoy Valley Common name: Chontara Kamsa name: Beuntajajch or Bebunjaja It is a well-known vine among the healers of the valley; it serves different purposes. The bark is scraped, toasted and applied over injuries and wounds. 55 2.4. Discussion The four collection sites corresponded to different cultural settings and different habitats as well. These differences are reflected in the plant material used for medicinal purposes in these regions. Eighteen plant families are represented in the collection list. The Asteraceae was represented by four plant species, all of them collected in the Sibundoy Valley and well known in the ethno-medical literature. This probably reflects the interest the healers of the Sibundoy Valley have raised in western researchers. In Uruguay and Argentina, an infusion made of stems, leaves and flowers of Conyza bonariensis is orally administered and used as a hepato-protectant agent. It also has anti-diarrhetic, anti-helmintic, diuretic and anti-urecimic effects, and is used in the treatment of rheumatism, gonorrhea and dysentery (Gupta, 1995). Eupatorium glutinosum is also well known in Ecuador because of its reputation for the treatment of peptic ulcer, as a cicatrizant of wounds and ulcers and in the treatment of diarrhea. A decoction of the leaves in water is used and orally administered, or else fresh leaves are crushed and directly applied onto the swollen or ulcerated areas. Eupatorium species have been traditionally used in different parts of the world, although some species are known to have a number i of adverse interactions in livestock and humans. It is important to bear in mind the fact there is a great variation in the chemistry of the natural products in the different species of Eupatorium (Sharma et al., 1998). 56 A. artemisioides is also a plant with a wide spectrum of medicinal uses. A leaf infusion is used to correct irregularities of the menstrual cycle and in treatment of dysentery, fresh leaves lightly roasted are crushed and rubbed on joints to alleviate the symptoms of rheumatism; an infusion of fresh dried flowers is used as a vermifuge; in Colombia a decoction of the leaves and inflorescences is used as an contraceptive. In cases of hematoma, the leaves are crushed and applied over the area as an anti-inflammatory and analgesic (Gupta, 1995). Another well known plant is Senna reticulata whose flowered stems are used to decorate the table of the "jaibana" (shaman) during the "chant of the jai" or the "spirits invocation". An infusion of the leaves is used as a laxative (Caballero, 1995). In Ecuador an infusion of the leaves of duglans neotropica is used as an astringent and against diarrhea, particularly in children; it is also used as an antiseptic to wash wounds and ulcers, for vaginal douches in cases of leukorrhea. An infusion of powdered bark of the fruit is used as a purgative (Gupta, 1995). Fresh latex of Vismia angusta and Vismia ferruginea is used commonly in Colombia and Peru to treat wounds and infected sores (Schultes, 1983). In Nicaragua the leaf of V. macrophylla is used to treat skin infection (Barrett, 1994). Ortiz mentions the use of this plant amongst the Sikuani and Cuiba in the treatment of gonorrhea, without specifying the useful part of the plant (Ortiz, 1985) 57 Symphonia globulifera is known as "mawinae" in Sikuani and the latex is used as an analgesic to be externally applied. A decoction of the bark is used in Gabon for scabies (Akendengue, and Louis, 1994) and in Panama the infusion is used to treat bloody vomiting (Joly et al., 1987). Interestingly, the benefits of Polygonum punctatum when the body is "watery" might be related to the use in traditional Chinese medicine of Polygonum hydropiper ("Water pepper" originary from India) or P. flaccidum which are used to remove dampness and food stagnancy (This terminology corresponds to traditional Chinese Medicine and correlation to western terminology is not evident.) (Huang, 1999). 58 2.5. References Akendengue, B. and Louis, A .M. , 1994. Medicinal plants used by the Masango people in Gabon. Journal of Ethnopharmacology 41: 193-200. Barrett, B., 1994. Medicinal Plants of Nicaragua's Atlantic Coast. Economic Botany 48: 8-20. Caballero, R., 1995. La etnobotanica en las comunidades negras e indigenas del delta del ho Patia. Ediciones Abya-Yala. Quito, p. 116. Candre H. and Echeverri, J.A. 1996. Cool Tobacco, Sweet Coca. Teachings of an Indian Sage from the Colombian Amazon. Themis Books. Foxhole, p. 267. Galeano, G. , 1992. Las palmas de la region de Araracuara. Tropenbos. Santafe de Bogota, p. 180. Gupta, M., 1995. 270 Plantas Medicinales Iberoamericanas. 1 s t Edition C Y T E D -SECAB,.Santafe de Bogota, p. 617. i Huang, K. C. 1999. The Pharmacology of Chinese Herbs. 2 n d Edition. C R C Press. Boca Raton.p. 512. 59 Instituto Geografico Agustin Codazzi (IGAC), 1996. Diccionario geografico de Colombia. Vol.4. IGAC. Santafe de Bogota, pp. 1764, 1574. Joly, L.G.; Guerra, S.; Septimo, R.; Solis, P.N.; Correa, M.; Gupta, M.; Levy, S. and Sandberg, F. 1987. Ethnobotanical inventory of medicinal plants used by the Guaymi Indians in Western Panama. Part I. Journal of Ethnopharmacology 20: 145-171. Motta, M.T., 1997. Participation comunitaria para manejo de bosques secundarios del Bajo Calima. CONIF. Santafe de Bogota, p. 181. Ortiz Gomez, F., 1985. Etno-botanica y Etno-zoologia guahibo Sikuani y Cuiba. Fundacion Segunda Expedition Botanica. Colciencias. Santafe de Bogota.p. 65. Sharma, O.P., Dawra, R.K., Kurade, N.P. and Sharma, P.D., 1998. A review of the toxicosis and biological properties of the genus Eupatorium. Natural Toxins 6: 1-14. Schultes, R.E., 1983. De Plantis Toxicaris e Mondo Novo Tropicale Commentationes X X X . Biodynamic Guttiferous Plants of the Northwest Amazon. Botanical Museum Leaflet Harvard University 29: 49-57. 60 Schul tes , R . E . and Raffauf, R .F . 1990. The Heal ing Forest : medic inal and toxic plants of the northwest A m a z o n i a . Edited by: Theodore R. Dudley. Dioscor ides P r e s s . Port land.p.484. \ i 61 Chapter 3 Biological Activities of Colombian Medicinal Plants "Every plant has a spirit of its own, a plant isn't just something else, it's a living thing. The spirit of a plant tells us /?ow to cure those who are sick; or shows us more plants. To call on that spirit we take yage." Inga H e a l e r M o c o a , C o l o m b i a 3.1. Introduction S k i n - r e l a t e d m o r b i d c o n d i t i o n s a r e n u m e r o u s a n d no t n e c e s s a r i l y c o n n e c t e d to a n in fec t i on . T h e r a t i ona l e is to a s s u m e that t he p lan t fo r w h i c h a n i n d i g e n o u s u s e i s r e p o r t e d wi l l h a v e s o m e k ind o f an t i b i o t i c and/or ant i v i ra l act i v i ty , h o p i n g that t he ex t r a c t wi l l b e d e t e c t e d a s h a v i n g e f f ec t o n v i r u s e s , b a c t e r i a o r y e a s t . T h i s c h a p t e r d e s c r i b e s t he b i o l o g i c a l s c r e e n i n g of c h e m i c a l e x t r a c t s of C o l o m b i a n m e d i c i n a l p l an t s w h o s e c o l l e c t i o n h a s b e e n d e s c r i b e d in c h a p t e r 2 . B e c a u s e o f the i r c u r r en t r e l e v a n c e , I b e c a m e pa r t i c u l a r l y i n t e r e s t e d in a n t i -v i r a l (e.g. h e r p e s s i m p l e x v i ru s ) a n d a n t i - C a n d i d a ac t i v i t i e s . Candida albicans is a n impo r t an t o p p o r t u n i s t i c p a t h o g e n c a u s i n g l o c a l o r s y s t e m i c i n f ec t i on s in p r e d i s p o s e d pa t i en t s w h o a r e i m m u n o l o g i c a l l y c o m p r o m i s e d o r u n d e r g o i n g p r o l o n g e d an t i b i o t i c t r ea tmen t . 62 C a n d i d o s i s h a s b e e n s h o w n to i n vo l ve a s p a r t i c p r o t e a s e s ( S A P ) s e c r e t e d f r o m Candida albicans a s a m a j o r v i r u l e n c e f a c t o r in C a n d i d a i n f e c t i on s ( H o e g l et al., 1996 ; D e B e r n a r d i s et al., 1999 ) . Inhibit ion of S A P h a s b e e n p r o p o s e d a s a n e w a p p r o a c h in the t r e a t m e n t o f th i s i n fec t i on . R e c e n t l y it h a s b e e n d e m o n s t r a t e d that H IV p r o t e a s e inh ib i tor s d e c r e a s e the a d h e r e n c e of f unga l c e l l s to ep i t he l i a l c e l l s in vitro ( B e k t i c et al., 2 001 ) . In v i e w of t he i nev i t ab l e d e l a y b e t w e e n the c o l l e c t i o n o f f r e s h ma te r i a l a n d the c h e m i c a l e x t r a c t i on , p lant m a t e r i a l c o l l e c t e d w a s a i r d r i e d in t he s h a d e at the r e s p e c t i v e c o l l e c t i o n s i t e s in o r d e r to a v o i d m i c r o b i a l c o n t a m i n a t i o n . M e t h a n o l w a s the s o l v e n t of c h o i c e in a p r e l im i na r y s c r e e n i n g b e c a u s e , in c o m p a r i s o n to o t h e r s o l v e n t s ( e thano l , c h l o r o f o r m , a c e t o n e , d i c h l o r o m e t h a n e ) , t h e quant i t y of c o m p o u n d s e x t r a c t e d is g r e a t e r (Eloff, 1998 ) . E l e c t r o n S p i n R e s o n a n c e S p e c t r o s c o p y ( E S R ) i s a l s o t e r m e d E l e c t r o n P a r a m a g n e t i c R e s o n a n c e ( E P R ) a n d it i s a t y p e o f s p e c t r o s c o p y s im i l a r to N u c l e a r M a g n e t i c R e s o n a n c e ( N M R ) . E S R a l l o w s s t u d y i n g c o m p o u n d s c o n t a i n i n g o n e or m o r e unpaired e l e c t r o n s . E S R s p e c t r a p a r a m e t e r s s u c h a s g -f a c t o r s a n d h y p e r f i n e sp l i t t ing c o n s t a n t s (Hf s ) p r o v i d e i n f o rma t i on a b o u t the s t r uc tu re of s o m e f r e e r a d i c a l s . T h i s t e c h n i q u e c a n b e a p p l i e d to t h e d e t e c t i o n a n d ident i f i ca t i on o f o r t ho a n d p a r a q u i n o l s o r q u i n o n e s in p l an t e x t r a c t s , w i thout pr ior i s o l a t i on . W h e n t h e s e c o m p o u n d s a r e in a l k a l i n e s o l u t i o n t h e y c o n v e r t to the i r c o r r e s p o n d i n g s e m i q u i n o n e r a d i c a l s w h i c h a r e s u b s e q u e n t l y o b s e r v e d by E S R ( P e d e r s e n , 2 0 0 0 ) . 63 This technique can be used then to identify compounds in crude extracts allowing selecting a smaller sub-population of crude extracts before engaging in a phytochemical analysis. In this chapter, I will illustrate an application of this technique in the identification of a compound (juglone) in a plant extract that exhibited activity against C. albicans \ \ \ 3.2. Materials and Methods 3.2.1. Microorganisms Viruses used in the antiviral assays were the enveloped double-stranded DNA herpes simplex virus (HSV-1) type 1 and the non-enveloped single-stranded RNA poliovirus (type 1 vaccine strain). Laboratory strains of bacteria and fungi were obtained from the University of British Columbia collection. Eight species of bacteria and one species of fungus were used in the screening process. The bacterial strains consisted of Gram- positive Bacillus subtilis, Streptococcus faecalis and Staphylococcus aureus K147 MS (methicillin-sensitive), while Gram-negative strains consisted of Escherichia coli DC 10, Klebsiella pneumoniae, wild type Pseudomonas aeruginosa H187 (wild), Salmonella typhimurium TA98, and acid fast Mycobacterium phlei. The fungus used was the yeast Candida albicans (UBC54). i An inoculum of each bacterial strain was suspended in 5 mL Mueller-Hinton broth (BBL) and incubated overnight at 37°C. The overnight cultures were diluted 1/10 with Mueller-Hinton broth (BBL™) (Becton, Dickinson and Company) before use. 64 The fungal cultures were prepared by swabbing the plate with a cotton swab and then transferring this to a vial containing 5 mL of Saboraud dextrose broth (BBL™) (Becton, Dickinson and company). 3.2.2. Preparation of Extracts Dried plant material was grounded manually. 20g of each ground plant were soaked in 200 mL methanol A . C . S . grade for 48 hours at room temperature. The extract was filtered through a Buchner funnel with Whatman No 1 filter paper and the residue was washed with methanol. The filtrate was concentrated and the remaining solvent was further removed under reduced pressure and freeze-dried for 24 hours. The yields of prepared extracts are shown in Table 3.1. 3.2.3. Antiviral Assays. Determination of Minimal Inhibitory Concentration (MIC) The antiviral assays were performed using the enveloped double stranded DNA herpes simplex virus (HSV-1) type 1 and the non-enveloped single strand RNA poliovirus (type 1 vaccine strain). Vero cells (African green monkey kidney cell line - American Type Culture Collection) were grown in monolayers in a 5% C 0 2 and 95% air atmosphere at 37°C, in Dulbecco's modified Eagle medium (MEM) (GIBCO-Life Sciences, Ontario) and 25 pg/mL gentamycin sulphate (Sigma), in 96-well microtest trays (Falcon) (Anani et al., 2000). When cells formed confluent monolayers, they were used for the assays. A solution of 40 mg/mL of each methanolic extract was 65 prepared and filtered through a sterile syringe filter of 0.2 pm pore diameter. Each extract was diluted 1:100 in M E M (Hudson et al., 1994) and serial two-fold dilutions of the extract were made (in duplicate) in M E M across a row of cells in an empty 96-well microtest tray (final concentration of methanol < 1%). The diluted extracts were transferred to the aspirated Vero cell monolayers previously prepared. \ These cultures were incubated at 37 °C for 60 min and examined microscopically for possible immediate cytotoxic effects. Then, 100 pL of virus corresponding to one hundred plaque-forming units (PFU) was added to each well. The tray was transferred to an environmental chamber (37 °C) and exposed to a combination of visible light plus UVA (long-wave ultraviolet) for 30 min with continuous gentle shaking (100rpm) of the tray in order to ensure homogeneity in the well. The fluorescent and black light blue lamps (BLB) were arranged to give approximately 5 watts/m 2 incident radiation of both visible and UVA light. Following the light exposure, the trays were returned to the cell culture incubator. Cultures were inspected periodically for viral cytopathic effects (CPE). In the case of HSV, complete cell destruction (100% viral C P E ) required 4 days, while for the poliovirus, 2 days. Absence of C P E was interpreted as positive results for antiviral activity. Partial inhibition was considered to be a negative result. The MIC was recorded as the minimum concentration of extract, which gave complete inactivation of virus infectivity (i.e., absence of viral CPE) . A solution of 66 methanol in M E M was used as a negative control and antiviral tests were repeated twice. 3.2.4. Cytotoxicity Assays To test for cytotoxicity, Vero cell monolayers were grown in 96 well microtiter plates (Falcon), and exposed to serial dilutions of the extracts, starting \ at a final concentration of 20 pg/mL of crude plant extract. The treated cells were then incubated at 37°C for 1 hour, exposed to UV-A light and visible light for 30 min and then re-incubated for 24 hours. The cells were examined microscopically for periodic assessment of changes in cell morphology or visible toxic effects (obvious cellular damage or lysis). A solution of methanol in M E M was used as a negative control (final concentration: 1%). Cytotoxicity assays were repeated twice. 3.2.5. Antimicrobial and Antifungal Assays The disk diffusion assay (Taylor et al., 1995) was used to screen for antimicrobial and antifungal activities. Samples were tested in duplicate; one in the dark and one exposed to UVA. One hundred microlitres of the diluted culture were spread on sterile Muller-Hinton agar (BBL™) (Becton, Dickinson and company) plates (for bacteria) or sterile Saboraud dextrose agar (BBL™) (Becton, Dickinson and company) plates (for fungi). Sterile paper discs were impregnated with 20 pL of a solution prepared with 100 mg of extract in 1 mL of methanol, and allowed to dry at room 67 temperature. The impregnated discs were placed on the plates and incubated for 30 min. to allow for diffusion. Gentamycin TO mg/ml (for bacteria) or nystatin 5mg/ml (for fungi) was used as a positive control. 8-Methoxypsoralen 1 mg/ml (8-MOP) was used as a positive control for light-activated extracts. Methanol was used as a negative control. To test for light activated antimicrobial/antifungal activity, one replicate plate was exposed to ultraviolet UVA light (5 W/m 2 , 320-400 nm from four Sylvania F20T12-BLB lamps) for two hours while the other was kept in the dark (Taylor ef al., 1995). The plates were incubated for 18 h (48 h for Mycobacterium phlei) before the resulting zones of inhibition were observed and recorded. Tests were repeated twice. 3.2.6. SAP Inhibition Screening The method described by Li and co-workers (2001) was modified to be faster and amenable in an automatic microtiterplate reader (Spectrafluor plus, T E C A N GmbH, Salzburg Austria). 3.2.6.1. Induction of SAP Production The procedure of Capobianco and co-workers (1992) was followed. Briefly, Candida albicans was grown overnight (15 hrs) in a shaker at 50 rpm and 35 0 C in Sabouraud dextrose media broth. The cells were centrifuged for 10 minutes at 17,300 rcf. The pellet was washed once with 10mM phosphate-buffered saline (PBS), pH = 7.0, centrifuged for 10 minutes at 17,300 rcf and re-68 suspended in 15 mL of filter-sterilized inducing medium (0.2% yeast extract, 0.2% bovine albumin, 2.0% dextrose), and incubated for 14 hours at 35 °C . The SAP-rich solution was centrifuged at 23,500 rcf for 3 minutes, the supernatant was collected, sterilized by filtration (0.2 micron filter from Fisher), and 1 mL aliquots from each fraction were stored at - 8 0 0 C until needed. 3.2.6.2. Inhibitory Activity against Secreted Aspartic Proteases. 3.2.6.2.1. Determination of the Optimal Substrate and SAP Extract Concentrations To determine optimum substrate and S A P extract concentrations an experiment, in which S A P extract and substrate concentrations were varied, was performed in a 96 well microplate. Immediately after adding the renin substrate (Molecular Probes R-2931 Eugene OR) the fluorescence at 360 ex/535 em was measured and fluorescence readings were taken automatically every 10 minutes for the next two hours. The increase in fluorescence during the incubation was determined by subtracting the fluorescence reading from the reading of a blank that contained citrate buffer instead of S A P extract. Optimum concentrations were chosen based on dose response curves and selecting a linear segment of the curve. The selected final concentrations were 40 uM renin substrate and 1/40 dilution of the S A P extract. 69 3.2.6.2.2. SAP Inhibition Assay for Crude Extracts A microtiter plate reader (Spectrafluor plus, T E C A N GmbH, Salzburg Austria) equipped with an excitation filter at 360 nm (35 nm bandwidth) and an emission filter at 535 nm (25 nm bandwidth) using 96 well microtiter plates (Falcon). Prior to each set \of measurements an automatic fluorescence gain adjustment was performed for the range of fluorescence changes expected. The integration time for the fluorescence signals was 40 ps without lag time at 15 flashes per well. The optimum gain setting of the instrument varied between 130-141. For measurements a microtiter plate was loaded with 50 pL of compound solution for a final concentration of 40 pM in sodium citrate buffer (methanol concentration < 2.0 %) followed by 50 pL of S A P extract (1/40 dilution). The mixture was incubated during one hour at 36 0 C in a shaker at 271 rpm. After the incubation time 100 pL of renin substrate was quickly added to obtain final concentrations of 40 pM. The final volume per well was 200 pL. Plant crude methanolic extracts were tested initially at a final concentration of 50 pg/mL in order to identify extracts with inhibition activity greater than 80%. The fluorescence signals of these extracts were recorded at 37 0 C during 20 min. for 10 cycles with a kinetic interval of 120 seconds. In principle, it is not essential to monitor the time course of fluorescence changes for each experiment. However, it should be visualised at least once to estimate the minimum time required for the reactions to be completed or whether or not there are drifts in the fluorescence signal during the time course of the 70 experiment. Tests were performed three times for each crude extract. Calculations of non linear regression curves were conducted with Excel (Microsoft) which is compatible with T E C A N software. A blank was used to correct the A R F U values for non enzymatic renin substrate degradation during the time course of the experiment. 3.2.7. Electron Spin Resonance (ESR) Twenty microliters of an Ethanol-Water (4:1) extract of Juglans neotropica was mixed with five microliters of NaOH (0.2M) and the mixture was shaken in the air. Ten microliters of the mixture in a capillary was introduced into the magnetic resonance cavity and E S R spectra was recorded according to the procedure described by Pedersen (2000). 3.3. Results 3.3.1. Antiviral Activity Thirteen extracts of the twenty-four species tested exhibited activity against HSV but none of them was active against poliovirus (Table 3.1). Extracts prepared from Byrsonima verbascifolia (Malpighiaceae), Iryanthera megistophylla, Vismia macrophylla (Clusiaceae) and Eschweilera rufifolia (Lecythidaceae) exhibited particularly good activity against HSV, comparable to potent extracts from previous studies (Anani et al., 2000). The minimal concentration causing cytotoxicity is reported in Table 3.1. 71 Extracts from Tagetes erecta (Asteraceae), Vismia macrophylla (Clusiaceae) and Picrolemna sprucei (Simaroubaceae) were found to be cytotoxic. Because of the cell damage produced by these extracts, it was not possible to determine antiviral activity. The results of the active extracts are listed in Table 3.1. The minimal inhibitory concentrations ranged from 2.5 to 25 ug/mL. \ 3.3.2. Antimicrobial Activity Only Tagetes erecta (Asteraceae) extract displayed photoactivity against Pseudomonas aeruginosa and Bacillus subtilis. None of the extracts was active against the Gram-negative bacteria tested, namely Escherichia coli, Salmonella typhimurium or Klebsiella pneumoniae. The extracts with the broadest spectrum of activity were: Adiantum latifolium (Pteridaceae); Eupatorium glutinosum (Asteraceae); Tagetes erecta (Asteraceae); Vismia macrophylla (Clusiaceae); Juglans neotropica (Juglandaceae); Eschweilera rufifolia (Lecythidaceae); Senna reticulata (Fabaceae); Symphonia globulifera (Clusiaceae).; Byrsonima verbascifolia (Malpighiaceae) ; Iryanthera megistophylla (Myristicaceae); Siparuna guianensis (Monimiaceae); Virola multinervia (Myristicaceae) ; Myrteola nummularia (Myrtaceae); Piper lanceaefolium (Piperaceae).; Polygonum punctatum i (Polygonaceae); and Duroia hirsuta (Rubiaceae). Twenty-two of the extracts showed activity against bacteria and two showed activity against Candida albicans (Table 3.2). Anti-Candida activity was observed with Piper lanceaefolium (Piperaceae) and Juglans neotropica (Juglandaceae). 72 3.3.3. Inhibitory Activity against Secreted Aspartic Proteases Sixteen of the extracts showed complete inhibition in the aspartic protease inhibition assay (Table 3.3). These were from the following species: Hydrocotyle umbellata (APIACEAE); Eupatorium glutinosum (Asteraceae); Symphonia globulifera (Clusiaceae); duglans neotropica (Juglandaceae); Eschweilera rufifolia (Lecythidaceae); Byrsonima verbascifolia (Malpighiaceae); Iryanthera megistophylla, Iryanthera tricornis and V7ro/a multinervia (Myristicaceae); Siparuna guianensis (Monimiaceae); Myrteola nummularia (Myrtaceae); Piper lanceaefolium (Piperaceae); Polygonum punctatum (Polygonaceae); Duroia hirsuta (Rubiaceae) and Solanum sp (Solanaceae). 73 Table 3.1 Antiviral Activity of Extracts of Selected Colombian Medicinal Plants Family / Species Part 3 Yield 0 (%) Cytotoxicity0 pg/mL H S V d ug/mL Poliovirus pg/mL A P I A C E A E Hydrocotyle umbellata A E 8.66 _ e -A S T E R A C E A E Ambrosia artemisoides LF 11.97 -Conyza bonariensis A E 9.82 — Eupatorium glutinosum LF 14.59 — Tagetes erecta FL 20.93 11.5 NFf NF C E L A S T R A C E A E Goupia glabra LF 18.02 — C L U S I A C E A E Vismia macrophylla BK 9.15 2.1 NF NF R E 35.97 5.5 Symphonia globulifera BK 8.20 25 74 J U G L A N D A C E A E Juglans neotropica BK 12.56 -— LECYTHIDACEAE Eschweilera rufifolia BK 10.23 - 8 F A B A C E A E Senna reticulata 16.03 -— MALPHIGIACEAE Byrsonima verbascifolia RB 11.48 6.5 — LF 18.59 2.5 MONIMIACEAE Siparuna guianensis LF 15.74 MYRISTICACEAE Virola multinervia R E 2.4 11.5 — BK 13.05 - 17 Iryanthera megistophylla BK 18.43 - 10 — Iryanthera tricornis LF 14.87 M Y R T A C E A E Myrteola nummularia A E 16.21 10.5 — P I P E R A C E A E Piper lanceaefolium LF 20.79 -P O L Y G O N A C E A E Polygonum punctatum A E 15.12 20 PTERIDACEAE 75 Adiantum latifolium A E 15.35 - 11.5 -R H A M N A C E A E Ampelozizyphus amazonicus LF 15.17 — 22 RUBIACEAE Duroia hirsuta LF 11.56 10.5 S I M A R O U B A C E A E Picrolemna sprucei LF 25.06 2.5 NF NF S O L A N A C E A E Solanum sp. BK 10.90 a Part extracted: A E , aerial parts; LF, leaves; BK, bark; RB , root bark; RT, root; R E , resin; FL. flowers b Weight (g) of crude methanolic extract per 100 g of dried plant material c Minimum concentration causing effect d Minimum concentration causing complete inhibition (MIC) of viral (HSV and Poliovirus) C P E . e Non active extract f Non feasible test 76 78 1 1 • i 1 1 i i i 1 1 1 1 I i i i i 1 1 1 i .+ +• .+ +" +_ "+ +" \ "+ +_ "+ +_ +• +_ ^F .+ +" .+ "+ +^  ^F i +_ "+ +^  +" +_ "+ +^  +• +^  +• .+ ^F ^F .+ ^F .+ +" +_ "+ i i +_ +• +_ +^  "+ .+ +" +^  "+ • +^  •"+ i + ^F +_ +• + ^F +_ +• 1 • • i • i i 1 - f 1 • i i i • i i 1 i i i 1 1 i i • l +_ +" i i AE C O L L _ J L U CL* C O L U < L L 1 L U < X Siparuna guianensis 1X1 < LU o < o 1-co cr: >--2 "5, • c Q . o CO & E CD 1 CO E o o CD C 1 CO CD "3 E CO s 5 L U < L U O < I-OC >-CD C •2 E E c TO o CD ! L U < L U O < cr: L U C L Q _ CD CD CD O C -2 CD . Q _ C L , L U < L U o < o o > o C L E .2 o c E c o § o C L < 1->-I C L o Q cn L U H C L E s •2 E C CD L U < L U O < < X err 79 1 1 • • • 1 1 1 1 i • • • +_ +• i +_ "+ i i .+ +• i i .+ +" i i +^  +• • • • i i +_ +• • • • i i 1 • i i 1 i i i i 1 1 i 1 I LF LL _J LL 1 Ampelozizyphus amazonicus UJ < LU o < m ID cc iS -£ CD s Q LU < LU O < CQ ID o cc < c o o 5 & CD E E CD 8 cj Ql LU < LU O < < _ l O c o cl CO E c •55 o CO c CO 'co CU > *co o Q . CD > o CD O CD x: c CD C o N O c CO CD c CO CD i _ LU CC o o CC L _ CD -O -*—» O O i _ cd" cc x ^ "+*" co" - CD • - > 'P CO "CO u_" I " TO CD CD C c o CO ro CL CD CD LU < TJ CD -i—» O ro i— x CD ro co o cj o cj .8 f CO o LU CD" .CD £ o E CD C Q . CtJ ro CD CO -Q CD ci co" c CD O 1 5 CD • 5 c CD O CD CJ CO E CO ' c ro c o CO CD c o E • § CD CO ^ o oo o r~~ .2 ro LU CD CO cj CJ o cj o i s •c Q . -2 CO CO ro CO .co" -Q CO CO o CD CQ ro CQ ] 5 Q . E 3 CD CJ CD -Q O O "= 2 d . o • .CO "CD o CD 3 . E § -SS "CD c o . E CD CO -a CO 2 oo CD CO o-C 5 CD CD ro •a CD g > ro 3 Q > 3.3.4. Inhibition of Secreted Aspartic Proteases Sixteen extracts presented complete inhibition in the enzymatic assay for secreted aspartic proteases. Only two extracts did not exhibit inhibition. Inhibition percentages are presented in Table 3 . 3 . \ 81 Table 3.3 Inhibition of Secreted Aspartic Proteases by Colombian Medicinal Plant Extracts Plant species Part Inhibition (%) A P I A C E A E Hydrocotyle umbellata A E 100 A S T E R A C E A E Ambrosia artemisoides LF 0 Conyza bonariensis A E 22 Eupatorium glutinosum LF 100 Tagetes erecta LF 34 C E L A S T R A C E A E Goupia glabra LF 0 C L U S I A C E A E Vismia macrophylla BK 62 Symphonia globulifera BK 100 J U G L A N D A C E A E Juglans neotropica BK 100 L E C Y T H I D A C E A E Eschweilera rufifolia BK 100 F A B A C E A E Senna reticulata LF 69.5 MALPHIGIACEAE Byrsonima verbascifolia RB 100 LF 100 MONIMIACEAE Siparuna guianensis LF 100 MYRIST ICACEAE Iryanthera megistophylla BK 100 Iryanthera tricornis LF 100 Virola multinervia BK 100 82 Table 3.3 (Cont.) Inhibition of Secreted Aspartic Proteases by Colombian Medicinal Plant Extracts Plant species Part Inhibition (%) M Y R T A C E A E Myrteola nummularia A E 100 P I P E R A C E A E Piper lanceaefolium LF 100 P O L Y G O N A C E A E Polygonum punctatum A E 100 PTERIDACEAE Adiantum latifolium A E 75 R H A M N A C E A E Ampelozizyphus amazonicus LF 34 RUBIACEAE Duroia hirsuta LF 100 S I M A R O U B A C E A E Picrolemna sprucei LF 13.5 S O L A N A C E A E Solanum sp. BK 100 83 3.3.5. Electron Spin Resonance Spectroscopy for Juglans neotropica extract. A spectrum of an ethanol-water extract of Juglans neotropica is shown in Figure 3.1. The g value and the proton hyperfine splittings (Table 3.4) are indistinguishable from those of an authentic juglone semiquinone spectrum. 84 85 OH O -Hyperfine Splitting Constants (Hfs) a 2 a 3 a 6 a 7 a 8 a O H 3.30 3.05 1.27 0.68 1.27 0.30 2.00429 g - value Table 3.4 Hyperfine Splitting Constants (a values) (in Gauss) and g values of juglone found in Juglans neotropica Extract i 86 3.4. Discussion Some of the extracts tested appear to be more potent than many of the antiviral extracts from plants examined to date (e.g. Taylor et al., 1996; Anani et al., 2000). The leaf extract of Byrsonima verbascifolia presented an inhibitory concentration as low as 2.5 pg/mL which is similar to the best activities found in extracts of seaweeds from British Columbia (Hudson et al., 1999). Byrsonima crassifolia (L.) H.B.K. is a tropical tree widely distributed in Mexico, Central and South America. It has been medicinally used since pre-hispanic times (Martinez-Vasquez et al., 1999), and antimicrobial and antifungal activity from bark and leaves has been widely reported (Martinez-Vazquez et al., 1999; Caceres et al., 1993). However, no bioassay-guided chemical fractionation has been carried out. Although other members of the genus Vismia have been studied extensively, little is known about the species reported here. The related species Vismia cayennensis exhibited HIV-inhibitory activity due to the presence of vismiophenone D (Fuller et al., 1999). A structurally related group of compounds collectively named guttiferones (prenylated benzophenone derivatives) were found to be responsible for the HIV-inhibitory activity in an extract of Symphonia globulifera (Gustafson et al., 1992). However, these compounds were also cytotoxic to the host cells used in the antiviral assay (Fuller et al., 1999; Gustafson et al., 1992). We detected cytotoxicity against Vera cells for the bark of Vismia macrophylla but no cytotoxicity was observed for its resin or in the bark of Symphonia globulifera. Further, two cytotoxic xanthones with isoprenoid groups have been reported from the root bark of S. globulifera (Nkengfack et al., 87 2002). On the other hand it is interesting to note that Symphonia globulifera and Iryanthera megistophylla, traditionally used to treat cutaneous leishmaniasis, displayed activity against HSV in our study. The potent anti-HSV activity (complete virus inactivation at 20 pg/mL) found in the methanolic extract of Polygonum punctatum contrasts with the results of Kott and coworkers (1999), who reported that the aqueous extract of the leaves of this species exhibited activity at a much higher concentration (EC 50= 169.7 pg/mL). The different conditions used in the extraction procedure might explain in part the difference between these two concentration values. Alternatively, the difference may be due to a lower concentration of bioactive compound(s) in the plant material collected by Kott and to the presence of photoactive antivirals. Leaf extract of Eupatorium glutinosum presented activity against Mycobacterium phlei, Bacillus subtilis and Staphylococcus aureus. This activity might be associated with the presence of labdene diterpenes, which have been shown to have weak antimicrobial activities against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. According to El-Seedi and coworkers (2002) this could support the vernacular medicinal use of E. glutinosum as an antimicrobial. i Arguably the most important of the extracts which I studied are the root bark and the leaf extracts of Byrsonima verbascifolia (see Table 3.1). However, the scarcity of the plant and the difficulty in obtaining adequate amounts made me discard this plant for future work. 88 Taking the anti-microbial and anti-viral results together, the plant extracts worth further study are: Eschweilera rufifolia, Iryanthera megistophylla, Virola multinervia, Myrteola nummularia and Duroia hirsuta. These extracts also exhibited complete inhibition in the secreted aspartic proteases inhibition assay. Based on the anti-Candida assays and the aspartic protease inhibition tests, the most promising extracts are Piper lanceaefolium and Juglans neotropica. The extract of Juglans neotropica was analysed by E S R and although only the semiquinone nucleus is identified by this procedure for some compounds like juglone, this gives an absolute proof of identity. Juglans species, and the compound juglone, have shown very good activity against dermatophytes (Ali-Shtayeh and Suheil, 1999). The presence of juglone detected by E S R may in part explain the wide range of antibiotic activity exhibited by Juglans neotropica bark extract. Based on these results and plant material availability P. lanceaefolium and /. megistophylla were selected for future phyto-chemical analysis along with associated biological activities. In the next two following chapters I will describe the work accomplished for each of these plant extracts. 89 3.5. Conc lus ion T h e s e e x p e r i m e n t s h a v e r e v e a l e d s o m e b i o l o g i c a l a c t i v i t i e s in C o l o m b i a n m e d i c i n a l p l an t s that c o u l d a c c o u n t fo r the i r t rad i t i ona l u s e s a s h e r b a l m e d i c i n e s . N o n e t h e l e s s t he a s s u m p t i o n that p a t h o g e n i c a g e n t s p r o d u c e the s k i n d i s e a s e s m i g h t not a l w a y s b e v a l i d . In o r d e r to c o n f i r m a p l an t u s e t he s c o p e o f t he a n a l y s i s h a s to b e b r o a d e r t h a n is p r e s e n t e d he re . T h i s is pa r t i cu l a r l y t rue w h e n o n e c o n s i d e r s t he f ac t that s o m e t rad i t i ona l u s e s a r e d i s r e g a r d e d w h e n e v e r t hey c a n n o t b e a p p r e h e n d e d in w e s t e r n t e r m s a n d i n s t e a d a n a s s u m p t i o n a b o u t a p a t h o g e n i c c a u s e is m a d e . In o the r w o r d s t h e r e i s a t e n d e n c y to put a s i d e c o n c e p t s a b o u t d i s e a s e w h o s e e p i s t e m o l o g y i s o u t s i d e t h e b o r d e r s o f o u r c a r t e s i a n ( l imited) u n d e r s t a n d i n g . S im i l a r l y , I b e l i e v e that the i n te rp l ay b e t w e e n s e c o n d a r y m e t a b o l i t e s f r o m t rad i t i ona l m e d i c i n a l p l an t s a n d the d i s e a s e d s t a te of t he ho s t m igh t b e fa r m o r e c o m p l e x t h a n m e r e l y t he resu l t o f a d i r e c t an t i b i o t i c ac t i v i t y e x e r t e d by a s i n g l e c h e m i c a l ent i ty. A l t h o u g h I h a v e p r o v i d e d e v i d e n c e o f an t i b i o t i c a n d ant iv i ra l act iv i ty in t h e s e p lant e x t r a c t s , it is o f g r e a t r e l e v a n c e to k e e p r e m i n d i n g o u r s e l v e s that p lant c h e m i c a l s h a v e p l e i o t r op i c e f f e c t s a n d t h u s t he f ac t o f f i nd ing i n te re s t ing an t i b i o t i c ac t i v i t i e s d o e s not c o n f i r m , ju s t i f y o r e x p l a i n the t rad i t i ona l u s e of a p lant. 90 3.6. REFERENCES Ali-Shtayeh, M.S. and Suheil, I.G. 1999. Antifungal activity of plant extracts against dermatophytes. Mycoses 42: 665-672. Anani, K., Hudson, J.B., de Souza, C , Akpagana, K., Towers, G.H.N., Arnason, J.T., Gbeassor, M. 2000. Investigation of medicinal plants of Togo for antiviral and antimicrobial activities. Pharmaceutical Biology 38: 40-45. Bektic, J . ; Lell, C. P.; Fuchs, A.; Stoiber, H.; Speth, C ; Lass-Florl; Borg-von Zepelin, M.; Dierich, M.P.; Wurzner, R. 2001. HIV protease inhibitors attenuate adherence of Candida albicans to epithelial cells in vitro. FEMS Immunology and Medical Microbiology 31: 65-71. Caceres, A.; Lopez, B.; Juarez, X. ; Delaguila, J . ; Garcia, S. 1993. Plants used in Guatemala for the treatment of dermatophytic infections. Evaluation of anti-fungal activity of 7 American plants, dournal of Ethnopharmacology 40: 207-213. Capobianco, J . O.; Lemer, C .G . ; Goldman, R.C. 1992. Application of a i fluorogenic substrate in the assay of proteolytic activity in the discovery of a potent inhibitor of Candida albicans aspartic proteinase. Analytical Biochemistry 204:96-102. 91 De Bernardis, F.; Arancia, S.; Morelli, L.; Hube, B.; Sanglard, D.; Schafer, W.; Cassone, A. 1999. Evidence that members of the secretory aspartyl proteinase gene family, in particular SAP 2, are virulence factors for Candida vaginitis. dournal of Infectious Diseases 179: 201-208. Eloff, J .N . 1998. Which extractant should be used for the screening and isolation of antimicrobial components from plants? dournal of Ethnopharmacology 60: 1-8. El-Seedi, H.R; Sata, N.; Torssell, K B.G. and Nishiyama, S. 2002. New labdene diterpenes from Eupatorium glutinosum. dournal of Natural Products 65: 728-729. Fuller, R.W., Westergaard, C.K., Collins, J.W., Cardellina II, J .H . , Boyd, M.R., 1999. Vismiaphenones D-G, new prenylated benzophenones from Vismia cayennensis. dournal of Natural Products 62: 67-69. Gustafson, K.R., Blunt, J.W., Monro, M.H.G., Fuller, R.W., Mc Dee, T.C., Cardellina II, J .H. , Mc Mahon, J .B. , Cragg, G.M. , Boyd, M.R., 1992. The guttiferones, HIV-inhibitory benzophenones from Symphonia globulifera, Garcinia livingstonei, Garcinia ovalifolia and Clusia rosea. Tetrahedron 48:10093-10102. 92 Hoegl, L ; Ollert, M.; Korting, H.C., 1996. The role of Candida albicans secreted aspartic proteinase in the development of candidoses. Journal of Molecular Medicine 74: 135-142. Hudson, J .B. , Graham, E.A., Towers, G.H.N. 1994. Antiviral assays on phytochemicals: The Influence of reaction parameters. Planta Medica 60, 329-332. Hudson, J.B. , Kim, J .H. , Lee, M.K., Hong, Y.K. and De Wreede R.E. 1999. Multiple antiviral activities in extracts of seaweeds from British Columbia. Pharmaceutical Biology 37: 300-306. Kott, V., Barbini, L., Cruanes, M., Munoz, J de D., Vivot, E., Cruanes, J . , Martino, V., Ferraro, G. , Cavallaro, L., Campos, R., 1999. Antiviral activity in Argentine medicinal plants, dournal of Ethnopharmacology 64: 79-84. Li, X .C . ; Jacob, M.R.; Pasco, D.S.; ElSohly, H.N.; Nimrod, A . C . ; Walker, L A . and Clark, A . M . 2001. Phenolic compounds from Micania myriantha inhibiting Candida Aspartic Proteases, dournal of Natural Products 64: 1282-1285. Martinez-Vasquez, M.; Gonzalez-Esquinca, A.R. ; Cazares Luna, L.; Moreno Gutierrez, M.N. and Garcia-Argaez, A . N . 1999. Antimicrobial activity of Byrsonima crassifolia (L.) H.B.K. dournal of Ethnopharmacology 66: 79-82. 93 Nkengfack, A .E . ; Mkounga, P.; Fomum, Z.T.; Meyer, M. and Bodo, B. 2002. Globulixanthones A and B, Two new cytotoxic xanthones with isoprenoid groups from the root bark of Symphonia globulifera. 2002. Journal of Natural Products 65: 734-736. Pedersen, J.A. 2000. Distribution and taxonomic implications of some phenolics in the family Lamiaceae determined by E S R spectroscopy. Biochemical Systematics and Ecology 28: 229-253. Taylor, R.S.L., Manandhar N.P., Towers G.H.N., 1995. Screening of selected medicinal plants of Nepal for antimicrobial activities. Journal of Ethnopharmacology 54: 153-159. Taylor R.S.L., Manandhar N.P., Hudson JB , Towers G.H.N. , 1996. Antiviral activities of Nepalese medicinal plants. Journal of Ethnopharmacology 52: 157-163. 94 Chapter 4 Antifungal Activity of Benzoic Acid Derivatives from Piper lanceaefolium Kunth "The day after the full, it is bad to bathe: If you do, the rainbow will urinate on you, \ And leave you covered with warts. For that, the children are to bathe indoors." Popular Kamsa saying cited in Mc Dowell (1989) 4.1. Introduction Based on the anti-Candida activity results and the inhibition of secreted aspartic proteases, a methanolic extract of P. lanceaefolium was chosen for further phyto-chemical analysis. Locally named in the Sibundoy Valley (southern Colombia) (see Fig. 4.1) as "cueche", a decoction of the leaves of Piper lanceaefolium Kunth (Piperaceae) is taken as a bath to treat morbid skin conditions. According to the information provided by Dona Clarita Buesaquillo and Don Miguel Chindoy Mutumbajoy these are malignant cutaneous manifestations whose occurrence is correlated with the drizzle, which usually accompanies the occurrence of a rainbow. This drizzle is thought to be the urine of the rainbow, otherwise known as "cueche". According to the literature, taking a bath outdoors after the full moon, would be the cause of getting the "rainbow's urine" (Mc Dowell, 1989). 95 Belief in the moon's influence on the human body is by no means exclusive to traditional societies. The apparent relationship between madness and moon is deeply rooted in the popular opinion that the word "lunatic" comes from the Latin word for moon, "Luna" (Zanchin, 2001). The moon's influence is believed to cause the rainbow to "urinate", source in turn of the referred morbid condition. In my study I assumed that it is caused by a pathogen (e.g. virus, fungus or bacterium). This rationale corresponds to the western pragmatic approach where the body is conceived as a machine and an infection as an extraneous agent that potentially affects the proper functioning of the body and consequently should be removed. P. lanceaefolium extract was selected because of its activity against C. albicans and complete inhibition of the secreted aspartic proteases enzymatic assay. This chapter reports the isolation and structural identification of the compounds present in an acetone extract of this plant, the minimal inhibitory concentrations for the active compounds and an assay to determine synergistic effects amongst such compounds. Since resistance against methicillin is such a problem in a clinical setting, the isolated compounds were tested against methicillin resistant Staphylococcus aureus. Species belonging to the genus Piper are used in medical systems world-wide. A number of species are used medicinally in Latin American folklore particularly in the western Amazon (Warrier et al., 1995; Schultes and Raffauf, 1992). The genus Piper (Piperaceae) has over 700 species distributed in both 96 hemispheres and its phytochemistry has been the subject of extensive review (Parmaref al., 1997). Except for a publication about P. lanceaefolium essential oils (Mundina et al., 2001) phytochemistry of this species has not been examined. 4.2. Materials and Methods 4.2.1. General Experimental Procedures Melting points (uncorrected) were recorded using a Gallenkamp melting point apparatus; optical rotations were measured on a J A S C O P-1010 polarimeter; IR spectra were obtained using KBr disks on a B O M E M MB-100 spectrophotometer; UV spectra were obtained with a Shimadzu UV 160U spectrophotometer; the NMR spectra were recorded on a Bruker AV-400 at 400 MHz (1H) and 100 MHz ( 1 3 C); multiplicity determinations (DEPT) and 2D NMR spectra (COSY, H M Q C , HMBC) were obtained using a Bruker AV-400 NMR spectrometer; high-resolution MS spectra were obtained on a Kratos MS 50 mass spectrometer; and TLC analysis was carried out on silica gel F254 plates (Merck). Preparative TLC was performed using silica gel 60 F254 (Merck) 250 micrometers thickness. The isolated compounds were visualized under UV at 254 nm, followed by development with ferric chloride spray reagent. 97 4.2.2. Plant Material L e a v e s o f P. lanceaefolium w e r e c o l l e c t e d in S i b u n d o y ( 01° 1 1 ' 0 0 " N, 0 7 6 ° 5 5 ' 0 0 " W ) ( D e p a r t m e n t of P u t u m a y o , C o l o m b i a ) ( S e e F i g . 4.1) a n d ident i f i ed by Dr. R. C a l l e j a s , A n t i o q u i a U n i v e r s i t y H e r b a r i u m (Mede l l i n , C o l o m b i a ) . A v o u c h e r s p e c i m e n ( L o p e z 5 6 ) w a s d e p o s i t e d in t h e h e r b a r i u m of t he S i n c h i Inst itute ( S a n t a F e d e B o g o t a , C o l o m b i a ) . 4.2.3. Extraction and Bioassay F r e s h l e a v e s w e r e a i r -d r i ed in t he s h a d e a n d g r o u n d . T h e p o w d e r ( 544 g) w a s e x t r a c t e d w i th 3 0 0 m L of a c e t o n e o n a s h a k e r f o r 2 5 m i n a n d the re su l t i ng e x t r a c t w a s c o n c e n t r a t e d to d r y n e s s to g i v e 37.8 g o f d r i e d m a t e r i a l . T h i s w a s d i s s o l v e d in m e t h a n o l a n d p a s s e d t h r ough a g l a s s c o l u m n c o n t a i n i n g C e l i t e 5 4 5 ( d i a t o m a c e o u s ea r th ) ( F i s he r ) . T h e re su l t i ng ex t r ac t w a s d i s s o l v e d in m e t h a n o l -w a t e r (3:1) a n d f r a c t i o n a t e d by l i qu id - l i qu id par t i t ion w i th h e x a n e , c h l o r o f o r m , e thy l ether , e t h y l a c e t a t e , b u t a n o l , a n d wa te r . T h e d i f f e ren t f r a c t i o n s w e r e t e s t e d a g a i n s t Candida albicans u s i n g a d i s k te s t a s s a y ( T a y l o r et al., 1995) . T h e an t i f unga l ac t i v i t y w a s l o c a l i s e d in the c h l o r o f o r m a n d e thy l e t h e r f r ac t i on s . T h e w a t e r s o l u b l e f r a c t i on d id not p r e s e n t act i v i ty a g a i n s t C. albicans. In a n a t tempt to f o l l ow the t rad i t i ona l p r e p a r a t i o n m e t h o d , 2 0 g of d r i e d l e a v e s w e r e e x t r a c t e d w i th 5 0 0 m L of bo i l i ng wate r , f i l te red ( W h a t m a n N o 1) a n d t he re su l t i n g ex t rac t w a s f r e e z e - d r i e d . T h i s e x t r a c t w a s fu r ther t e s t e d a g a i n s t t he d e r m a t o p h y t e Trichophyton mentagrophytes u s i n g a d i s k tes t a s s a y ( S e e S e c t i o n 3.2.5) w i th a n 98 incubation period of 72 h at 30°C. Amphotericin B (Sigma Chemical co.) was used as a positive control and methanol as the negative one. Figure 4.1 Map of the Sibundoy Valley (Colombia) i 99 4.2.4. Isolation of Compounds The liquid-liquid partition fractions (chloroform and ethyl ether) (22.3 g) were subjected to column chromatography over silica gel (346 g, silica gel 230-400 mesh, Merck) using a gradient of ethyl acetate in dichloromethane. A total of 68 fractions (230 mL each) were collected and combined into seven pools (l-VII) on the basis of similar thin layer chromatography (TLC) profiles. Pools I and IV, which were eluted with C H 2 C I 2 and C H 2 C I 2 / E 1 O A C (9:1) respectively, showed activity against Candida albicans. Crystallization of the different pools (except pool IV) produced compounds 1 (1.93 g), 2 (196 mg), 3 (120 mg), 4 (126 mg), 5 (3.06 g) and 6 (6 mg). Fractions in pool IV were combined and applied to a column of silica gel (70-230 mesh) and eluted using continuous gradients of ethylacetate in hexane (0-100%). The fraction that displayed activity against C. albicans was subjected to preparative TLC to yield 7 (53 mg). 4.2.5. Direct Bioautographic A s s a y . A bioautographic agar overlay assay was used to detect active fractions obtained during the chemical separation. Fractions of 700 pg each were spotted on chromatographic silica gel 60 T L C plates (Merck) and developed using CH 2 CI 2 -E tOAc (50:50) as solvent system. Nystatin (100 pg) was used as a positive control. After thorough drying for complete removal of the solvent, an inoculum of C. albicans {ca 10 7cells/mL) in molten agar (Saboraud broth medium agar, phenol red) was distributed over the chromatograms. The medium solidified 100 a s a th in l a ye r ( c a 1 m m l a y e r t h i c k n e s s ) , a n d the T L C p l a t e s w e r e i n c u b a t e d o ve r n i g h t at 3 7 0 C in a mo i s t a n d c h a m b e r e d e n v i r o n m e n t . T h e inh ib i t ion z o n e s w e r e v i s u a l i z e d by s p r a y i n g wi th a n a q u e o u s s o l u t i o n o f m e t h y l -t h i a z o l y t e t r a z o l i u m c h l o r i d e ( M T T ) (0.1 mg/mL). A c t i v e c o m p o u n d s w e r e d e t e c t e d a s c l e a r y e l l o w s p o t s a g a i n s t a pu rp l e b a c k g r o u n d . T w o add i t i ona l b i o a u t o g r a p h y T L C p l a t e s w e r e d e v e l o p e d a n d s p r a y e d w i th f e r r i c c h l o r i d e a n d van i l l in/su l fur ic a c i d to v i s u a l i z e the c h r o m a t o g r a p h i c p ro f i le o f p h e n o l i c c o m p o u n d s ( R a h a l i s o n etal., 1 994 ; S a x e n a etal., 1995 ) . 4.2.6. Minimum Inhibitory Concentration (MIC) Determination. M i n i m a l inh ib i tory c o n c e n t r a t i o n s v a l u e s w e r e d e t e r m i n e d by the broth d i lut ion m e t h o d u s i n g a c o n c e n t r a t i o n of 6.0 x 1 0 4 c o l o n y f o r m i n g un i t s/mL of Candida albicans g r o w n in S a b o u r a u d b ro th m e d i u m a n d 5.0 x 1 0 4 c o l o n y f o r m i n g un i t s/mL of Staphylococcus aureus M S in M u l l e r - H i n t o n b ro th m e d i u m . T h e a p p e a r a n c e of turb id i ty a f te r 2 4 h. d e t e r m i n e d the M I C , d e f i n e d a s the l o w e s t c o n c e n t r a t i o n of s u b s t a n c e that p r e v e n t e d g r owth . S o l u t i o n s o f te s t c o m p o u n d s w e r e p r e p a r e d in m e t h a n o l , a n d d i l u ted w i th S a b o r a u d m e d i u m a n d M u l l e r -H in ton m e d i u m to g i v e f ina l d i l u t i on s r a n g i n g f r o m 4 0 0 to 0.4 pg/mL. T h e f ina l c o n c e n t r a t i o n of m e t h a n o l in t he a s s a y d i d not e x c e e d 2 % . T h e a s s a y w a s c a r r i ed out in 9 6 - w e l l m ic ro t i te r p l a te s . I ncubat i on w a s at 3 7 0 C fo r 2 4 h. A m p h o t e r i c i n B w a s u s e d a s a po s i t i ve c on t r o l fo r y e a s t a n d g e n t a m y c i n for b a c t e r i a wi th a M I C v a l u e o f 0 . 055 a n d 0.1 pg/mL. r e s p e c t i v e l y ( R a h a l i s o n et al., 1994) . 101 4.2.7. Synergy Effects In order to study possib le synergist ic inhibitory activit ies against C. albicans, a microtiter assay w a s used (see Table 4.1). Different combinat ions of methanol ic solut ions were used and tested for activity against Candida albicans grown in Sabouraud broth medium (8.0 x 1 0 4 colony forming units/mL). The appearance of turbidity after 24 h. determined the inhibitory activity. Solut ions of test compounds were prepared in methanol , and diluted with Saboraud medium to give final concentrat ions of 100 pg/mL in wel ls for single compounds (1-7 shown in bold) and 50 pg/mL for wel ls with combinat ions of two compounds . The final concentrat ion of methanol in the a s s a y w a s 2 .5%. The a s s a y was carr ied out in 96-well microtiter plates incubated at 37°C for 24h. Amphoter ic in B was used as a posit ive control for yeast and Sabouraud broth medium with methanol (2.5%) as a negative control. Two repl icates were carried out. 7-1 7-2 7-3 7-4 7-5 7-6 7 6-1 6-2 6-3 6-4 6-5 6 5-1 5-2 5-3 5-4 5 4-1 4-2 4-3 4 3-1 3-2 3 2-1 2 1 i Table 4.1 Microtiter Plate Assay to Test for Synergy Effects (numbers inside the grid correspond to the compound identification number) 102 4.2.8. Secreted Aspartic Proteases Inhibition Assay for Isolated Compounds Isolated compounds were dissolved in sodium citrate buffer and tested at a final concentration of 40 pM. Reaction rates were determined as described in 3.2.6.2.1. The negative control was secreted aspartic proteases in the presence of renin substrate for a non inhibitory effect. These proteases in the presence of the inhibitor pepstatin (0.006 pg/mL) were used as a positive control (100% inhibition). Only compounds with activity equal or greater than 50% of the positive control were considered for further investigation. 4.3. Results 4.3.1. Plant Description The botanical description of Piper lanceofolium Kunth is as follows: Arborescent; branches nodose, silky; leaves lanceolate, 3-5 wide x 15-27 cm. Long, apex and more or less cuadately acuminate, base inequilaterally cordulate, with the lobes often overlapping, pinnately nerved from below the middle, the impressed nerves 8 or 9 on each side, sharply ascending, more or less finely rugose-bullate and silky-hairy above, at least along the nerves, lacunose and densely villous or subtomentose benneath, drying rather coriaceous, subopaque; petiole nearly suppressed, silky; spikes strongly curved, mostly 3 mm. thick x 8-12cm. Long, often mucronate; peduncle 10-20 mm. long, silky; bracts rounded-or triangular-subpeltate, marginally fringed; fruit suboblong-trigonous, glabrous; stigmas linear, 3 or 4, sessile (Trelease, 1950). 103 4.3.2. Chemical Isolation and Characterisation Column chromatography, followed by preparative TLC (see Experimental Section) of the chloroform and ethyl ether fractions, resulted in the isolation of four previously unknown prenylated benzoic acid derivatives (1-4) (Fig. 4.3; 4.4; 4.5; 4.7). Also known compounds such as taboganic acid (5) (See Figure 4.8) (Terreaux et al., 1998), pinocembrin (6) (See Figure 4.9) (Fukui et al., 1988), and pinocembrin chalcone (7) (See Figure 4.10) (Bremner and Meyer, 1998) were isolated for the first time in this plant, and identified by comparison of their physical and spectral data with those previously reported. Cyclolanceaefolic acid methyl ester (1): Obtained as an amorphous powder, mp 146-147 °C; [a ] 2 4 D =- 5.7° (c 0.5, MeOH);. IR (KBr) vmax 3253, 2984, 1713, 1664, 1608, 1587, 1456, 1432, 1394, 1373, 1286, 1229, 1176, 1097, 1008, 947, 929, 903, 771, 751 cm - 1 ; UV (MeOH) W (e) 240 (21 007), 334 (2 195), 203 (6 600); 1 H and 1 3 C NMR data, see Table 4.3; EIMS m/z 250 [M]+ (30), 235 (80), 219 (12), 194 (100), 163 (60), 135 (40), 108 (15), 91 (5), 79 (60), 77 (10), 51 (35); HREIMS m/z 250.08334 (calcd for C i 3 H 1 4 0 5 250.08412) i Cyclolanceaefolic acid (2): Isolated as white crystals, mp 244-246 °C; [ a ]24 D = _4.o° ( C 0.6, MeOH); IR (KBr) vmax 3233, 1683, 1675, 1607, 1587, 1488, 1373, 1292, 1175, 1092, 996, 927, 905, 880, 771, cm - 1 ; UV (MeOH) X m a x (s) 238 (19 920), 338 (2 714), 203 (7 241); 1 H and 1 3 C NMR data, see Table 4.3; EIMS 104 m/z: 236 [M] + (40), 221 (75), 219 (12), 180 (100), 152 (20), 135 (15), 79 (8), 51 (5); H R E I M S m/z 236.06836 (calcd for C 1 2 H 1 2 0 5 236.06847) . Lanceaefolic acid methyl ester (3). Obta ined a s yel lowish need les , mp 109-110 °C , [ a ] 2 4 D = -8.6° (c 0.1 M e O H ) ; IR (KBr) vmax 3459, 1723, 1713, 1638, 1588, 1482, 1438, 1317, 1231, 1186, 1142, 1093, 1003, 910, 857 , 771 , 751 cm" 1 ; U V ( M e O H ) W (e) 240 (21 086), 338 (2 525), 2 0 3 (4 634); 1 H and 1 3 C - N M R data s e e Tab le 4 .3 ; E I M S m/z: 250 [M] + (25), 235 (100), 219 (10), 194 (40), 173 (60), 135 (10), 108 (10), 83 (20), 55 (25); H R E I M S m/z 250 .08436 ( ca lcd . for C 1 3 H 1 4 0 5 250.08412) . Lanceaefolic acid (4): Obta ined a s orange-yel low need les (acetone): mp 253-255 °C; [a ] 2 4 D = -14 .3 ° (c 0.1, M e O H ) ; IR (KBr) vmax 3477 , 1713, 1688, 1638, 1584, 1482, 1422, 1313, 1283, 1134, 1096, 1057, 970 , 884, 770, 695 cm" 1 ; U V ( M e O H ) X m a x (e) 236 (19 562), 338 (2 898), 205 (8 326); 1 H and 1 3 C N M R data s e e Tab le 2; E I M S m/z 236[M]+ (30), 221 (100), 180 (55), 152 (10), 125 (10), 83 (15), 55 (15); H R E I M S m/z 236 .06832 (calcd for C i 2 H 1 2 0 5 , 236.06847) . Taboganic acid (5): Obta ined a s yel low crystals , mp 170-172 ° C E I M S m/z 220[M]+ (6), 205 (100), 165 (18). 1 H - N M R (400 M H z , ace tone -c / 6 ) : 8 8.58 (1H, d, J = 2.12 H z H-2), 8.13 (1H dd, d = 2.12, 8.74 H z , H-6), 7.10 (1H d d = 1.98 H z H-2'), 7.05 (1H d , d = 8.74 H z H-5), 2 . 2 5 a (3H, s , H-4'), 2 . 0 7 a (3H, s , H-5') 105 Pinocembrin (6): Obtained as pale yellow needles, mp 198-199 ° C EIMS m/z 256[M]+ (100), 238 (12), 179 (84), 152 (85), 124 (88), 105 (43), 104 (33), 103 (35), 77 (64), 69 (56). 1 H - N M R (400 MHz, acetone-cfe ,): 5 : 2.80 (1H, dd, J = 17.1, 3.15 Hz, H-3a), 3.16 (1H, dd, J = 17.1, 12.7 Hz, H-3b), 5.56 (1H dd, J = 3.13, 12.7 Hz, H-2), 5.95 (1H d, J = 2.2 Hz H-6), 5.97 (1H d , J = 2.2 Hz H-8), 7.38-7.60 (5H, m, H of ring\B). Pinocembrin chalcone (7): Obtained as yellow powder, EIMS m/z 256[M]+ (100), 238 (10), 179 (78), 152 (59), 124 (30), 105 (28), 104 (22), 103 (27), 77 (33), 69 (23). 1 H-NMR (400 MHz, acetone-d 6): 6 7.45 (2H, m, H-3, 5), 7.69 (2H, m, H-2,6), 7.75 (8-H d, J = 15.7), 8.23 (7-H d J = 15.7) 4.3.3. Biological Activity Growth inhibitory effects against Candida albicans were detected in lanceaefolic acid methyl ester (3) and pinocembrin chalcone (7) using disk diffusion and bioautographic assays (Terreaux et al., 1998) (See Figure 4.11). The aqueous extract of the leaves of P. lanceaefolium did not present activity against C. albicans nor against Trichophyton mentagrophytes. These compounds exhibited a minimal inhibitory concentration of 100 ug/mL for Candida albicans. Pinocembrin and its chalcone resulted also in a minimal inhibitory concentration of 100 ug/mL. for Staphylococcus aureus MS and none of the compounds presented at a concentration less than 320 ug/mL for S. aureus MR 106 Positive - • control: Nystatin Figure 4.2 Bio-autography with Candida albicans No synergy effects were detected for the combination of compounds used. Only compounds 3 and 7 presented activity against C. albicans and only their combination presented inhibitory activity. i 107 4.3.4. Aspartic Proteases Inhibition Assay None of the compounds caused 50% or more inhibition of aspartic protease at 40 pM. Consequently they were not selected for further characterisation (i.e. inhibition studies). Table 4.2 shows the inhibition percentages for the aspartic proteases assay. Table 4.2 Inhibition of Secreted Aspartic Proteases by compounds from P. lanceaefolium Compound Inhibition (%) 1 16.9 2 15.3 3 3.1 4 12.5 5 11.9 6 15.6 7 16.1 108 o I/O X uO oo o lO I lO CO CN 2 , CN 3, CT o CN CO ^ CM CD 2 , co C N Oi cd CD CO q CT) CO LO CN cp CD CD Oi 0 0 CN CJ) o CM C^O CN CD LO ^C0 3 cN in, LO CD CD CX) CD CT CN CJ) 0 0 CN CD Oi T3, CO o CN CO L— oo q 2 , CM o CN q CD CO CO o 3 -0 0 CD CM cd CN CM CM CN CM CM cr CM CO CO CN CM CM LO ~7n "co" o CN LO oo CN CM CO c g w o CL CN 0 0 ^3- LO CD CO O o CN -*-» "c/T CT 0 0 h- CD oo q CT> LO CO CT co CN CT) T — CM CN CM LO CT) CD CD CN o X o o lO oo CN X I/O CN 0 0 CM CN ;d CD O) CN CM CD 0 0 O q co CT 0 0 2 2 2 2 cn. oo 0 0 CM T — CT) LO q cq T - ' co CT) CN CD CD CN CM LO Oi CM CN CN LP CD X lO CO oo 0 0 CM CD . CT CN CN . CD CM CO 0 0 CD 0 0 CO c o •4—' o CL CM 0 0 <«fr CD LO CD 0 0 CD O O O CT 1 -o r o X o 4.4. Discussion 4.4.1. Structural Elucidation C o m p o u n d 1 was obtained as a white amorphous powder and had a molecular ion peak at m/z 250.08334 in H R E I M S suggest ing a molecular formula of C13HUO5. The IR spectrum indicated the presence of two carbonyl groups (1713, 1664 cm" 1 ) and an aromat ic ring (1608, 1587 cm ' 1 ) . The ass ignments of proton and carbons were obtained by analys is of its 2D N M R ( H M Q C and H M B C ) . The 1 H N M R spectrum (Table 4.3) exhibited two aromat ic proton resonances at 5 7.62 (1H, d, J = 2.1 Hz) and 7.96 (1H, d, J = 2.1 Hz) . On the bas is of chemica l shifts and coupl ing patterns, a 1, 2, 3, 5- tetrasubstituted phenyl ring was sugges ted to be present in the molecu le . In addit ion, the 1 H N M R spectrum displayed signals for one methylene group at 8 2.83, and three methyl groups at 5 1.48 (6H, s) and 3.86 (3H, s). The 1 3 C - N M R spect rum (Table 4.3) and D E P T exper iments of 1 showed 13 s ignals : three methyls, one methylene, two methines, and seven quaternary carbons, of which the s ignals at 8 152. 7, 123.4, 121.1, 119.2, 121.2, 147.9, 166.5, and 52 .3 , together with a sharp singlet (3H) at 8 3.86 in the 1 H N M R spectrum, implied the p resence of a methyl es ter of 3,4,5-trisubstituted benzo ic ac id . The s ignals at 8 191.77, 48 .92 , 81.75, and 26.47 (x2) together with the methylene resonance (singlet) and two aliphatic methyl groups of magnet ic equiva lence in the 1 H N M R spect rum, a s well as the H M B C (Figure 4.6) correlat ions between C -2 , C-4 and methylene (H-3) revealed the connectivity C O (4)-CH 2 (3)-C(2)-(CH 3 )2 • The H M B C spect rum of 1 a lso showed correlat ions from the carbonyl group at 8 191.77 to H-5, from the carboxyl group to H-5 and no H-7, and from C-8a to H-7 and H-5, which indicated that the ketone carbonyl and ester groups were attached at C-4a and C-6, respectively. Thus, the structure of 1 was determined to be cyclolanceaefolic acid methyl ester(methyl ester of 8 -hydroxy-2,2'-dimethyl-6-carboxy-chroman-4-one). \ Figure 4.3 Cyclolanceaefolic acid methyl ester ( 1 ) Compound 2 was isolated as white crystals. The HREIMS of this compound gave a molecular ion at m/z 236.06836 corresponding to a molecular formula of C 1 2 H i 2 0 5 . IR absorptions at 3233, and 1683 cm' 1 suggested the presence of hydroxyl and carbonyl groups. 1 H and 1 3 C - N M R data (Table 4.3) and DEPT experiments revealed 12 signals: two methyls, one methylene, two methines, and seven quaternary carbons. Compounds 1 and 2 were found to have similar structures by comparison of their NMR spectra. The observed difference was the loss of the methoxy signals ( 1 H-NMR at 5 3.86 and 1 3 C - N M R at 5 52.27) in the NMR spectrum of 1, which indicated that the methyl ester carbonyl was replaced i n in 2 b y a c a r b o x y l g roup . T h u s , the s t r uc tu re of 2 w a s a s s i g n e d a s c y c l o l a n c e a e f o l i c a c i d ( 8 - h y d r o x y - 2 , 2 ' - d i m e t h y l - 6 - c a r b o x y - c h r o m a n - 4 - o n e ) . Figure 4.4 Cyclolanceaefolic acid (2) C o m p o u n d 3 w a s o b t a i n e d a s y e l l o w i s h n e e d l e s . T h e H R E I M S i n d i c a t e d a m o l e c u l a r f o r m u l a o f C13H14O5. T h e IR s p e c t r u m i n d i c a t e d t w o c a r b o n y l g r o u p s a t 1 7 3 0 a n d 1 7 2 4 c m " 1 . T h e 1 H N M R s p e c t r u m c o n t a i n e d t w o a r o m a t i c p ro ton s , w i th t he c h e m i c a l sh i f t a n d sp l i t t ing pa t te rn t y p i c a l o f H-2 a n d H-6 of a 1,3,4,5- tet ra s ub s t i t u t ed b e n z e n e r ing. F u r t h e r m o r e t h e s i g n a l s at 8 196 .88 , 120 .23 a n d 161 .79 in t he 1 3 C - N M R s p e c t r u m a n d a s i g n a l at 8 7 .06 in t h e 1 H - N M R s p e c t r u m imp l i ed a c o n j u g a t e d k e t o n e s y s t e m . T h e s i g n a l s in t h e 1 H N M R s p e c t r u m at 8 2.10 ( 3 H , s ) , 2 .24 ( 3 H , s ) w e r e a s s i g n e d t o v i n y l m e t h y l g r o u p s p o s i t i o n e d o n a t r i subs t i tu ted d o u b l e b o n d . A s h a r p , 3 H s i n g l e t at 8 3.85, t o g e t h e r w i th the 1 3 C N M R r e s o n a n c e s a t 8 166.48, a n d 5 2 . 2 9 s u g g e s t e d t h e p r e s e n c e o f a m e t h y l 112 ester. Correlations in the HMBC spectrum from C-7 to H-2 and H-6, from C-8 to H-2, and from C-3 to H-9 suggested that the ester carbonyl and the ketone carbonyl were attached to C-1 and C-3 of the benzene ring, respectively. C O S Y , HMQC and HMBC NMR spectra permitted the complete assignment of all the protons and carbons as shown in Table 2. The above evidence led to the elucidation of structure 3 as lanceaefolic acid methyl ester [Methyl ester of 4,5-dihydroxy-3-(3-methyl-2-butenoyl) benzoic acid], which was confirmed by comparing the NMR data with the known compounds taboganic acid methyl ester (Roussis et al., 1990) and piperoic acid (Ampofo et al, 1987). Figure 4.5 Lanceaefolic acid methyl ester (3) 113 Figure 4.6 Selected HMBC correlations for 1 and 3. (Arrows denote HMBC correlations from C to H) C o m p o u n d 4 w a s o b t a i n e d a s o r a n g e - y e l l o w n e e d l e s . T h e H R E I M S s h o w e d a m o l e c u l a r ion at m/z 2 3 6 . 0 6 8 3 2 , s u g g e s t i n g a m o l e c u l a r f o r m u l a ion of C12H12O5. T h e p r e s e n c e of a 1,3,4,5- t e t r a s ub s t i t u t ed a r o m a t i c r ing in the s t ruc tu re b e c a m e c l e a r f r o m the c o n s i d e r a t i o n of r e s o n a n c e s a t t r i bu ted to the a r o m a t i c r i ng s in t he 1 H a n d 1 3 C N M R s p e c t r a . C o m p o u n d s 3 a n d 4 s h o w e d ve r y s im i l a r N M R s p e c t r a e x c e p t that the s i g n a l s o f the m e t h y l g r o u p at 5H 3.85 a n d 8c 52 .29 w e r e on l y p r e s e n t in t he s p e c t r a o f 3. T h u s , t he s t r u c t u r e o f l a n c e a e f o l i c a c i d [ 4 , 5 -d i h yd roxy - 3 - ( 3 -methy l - 2 -bu tenoy l ) b e n z o i c a c i d ] w a s a s s i g n e d a s 4. 114 Figure 4.7 Lanceaefolic acid (4) O H O Figure 4.8 Taboganic acid (5) 3-(1 '-Oxo-3'-methyl-2'-butenyl)-4-hydroxy-benzoic acid 115 OH 0 Figure 4.9 Pinocembrin (5,7-Dihydroxyflavanone) (6) Figure 4.10 Pinocembrin chalcone (7) Most of the chalcones isolated from the Piper species are oxygenated at the C-2', C-4' and C-6' positions and the B ring is generally unsubstitued (Parmar i et al., 1997). Pinocembrin chalcone, isolated for the first time in Piper species, follows this substitution pattern. On the other hand, 5,7-Dihydroxyflavanone, (pinocembrin) has been isolated in P. hostmannianum (Diaz et al., 1987). Taboganic acid was reported for the first time as a natural product in Piper 116 dilatatum L.C. R i c h ( T e r r e a u x et al., 1 998 ) a l t h o u g h its m e t h y l e s t e r h a d b e e n a l r e a d y i s o l a t e d f r o m Piper taboganum ( R o u s s i s et al., 1990 ) . 4.4.2 Biological Activity D e t e r m i n a t i o n o f t he m i n i m a l i nh ib i to ry c o n c e n t r a t i o n o f t he c o m p o u n d s c o n f i r m e d that l a n c e a e f o l i c a c i d m e t h y l e s t e r (3) a n d p i n o c e m b r i n c h a l c o n e (7) a r e at l e a s t part ly r e s p o n s i b l e f o r t h e an t i f unga l act i v i ty o f Piper lanceaefolium. A l s o it a p p e a r s that p i n o c e m b r i n a n d its c h a l c o n e a r e at l e a s t part ia l ly r e s p o n s i b l e fo r t he an t i bac te r i a l ac t i v i t y of l e a f e x t r a c t s . It is i n te re s t i ng to no te that o n l y p i n o c e m b r i n c h a l c o n e but not p i n o c e m b r i n , e x h i b i t e d act iv i ty a g a i n s t C. albicans. T h e r e is a l o s s o f act iv i ty b y c y c l i z a t i o n to t he c o r r e s p o n d i n g f l a v a n o n e . S i m i l a r l y o n l y l a n c e a e f o l i c a c i d m e t h y l e s t e r (3) bu t no t c y c l o l a n c e a e f o l i c a c i d m e t h y l e s t e r (1) p r e s e n t e d ac t i v i t y a g a i n s t C. albicans. It w o u l d a p p e a r that c y c l i z a t i o n i s c o r r e l a t e d w i t h t he l o s s o f act iv i ty. 3 O Figure 4.11 Chalcones (1,3-diaryl-2-propen1-ones) general skeleton 117 T s u c h i y a a n d c o - w o r k e r s ( 1 994 ) r e p o r t e d an t i - f unga l ac t i v i t y o f d i f fe rent s y n t h e t i c h y d r o x y - c h a l c o n e s a g a i n s t Candida s p e c i e s . T h e a u t h o r s s t a t e d that sub s t i t u t i on of the c h a l c o n e s wi th h y d r o x y l g r o u p s e n h a n c e s t he anti-Canc//c/a act iv i ty. T h i s w o u l d p r o v i d e both l i poph i l i c a n d h y d r o p h i l i c m o i e t i e s to the c h a l c o n e s . H o w e v e r , t he a u t h o r s repor t act i v i ty in c h a l c o n e A a n d n o n e in c h a l c o n e B that h a s a n add i t i o na l h y d r o x y l in 2 ' po s i t i on ( s e e F i g u r e 4.12). Interest ing ly , a s y n t he t i c p r e p a r a t i o n o f p i n o c e m b r i n c h a l c o n e d i s p l a y e d act iv i ty a g a i n s t C. glabrata but not C. albicans. T h e i m p o r t a n c e o f t h e d o u b l e b o n d w a s a l s o h i gh l i g h ted . Active Inactive Chalcone A Chalcone B Figure 4.12 Effect of 2' Hydroxyl Group Removal in Chalcones Antifungal Activity (Tsuchiya, 1994) 1 1 8 S i m i l a r l y S a t o a n d c o - w o r k e r s (1994) t e s t e d a g r o u p of s y n t he t i c c h a l c o n e s a g a i n s t Candida s p e c i e s . T h e y f o u n d that 2 ' - h y d r o x y l g r o u p w a s impo r tan t fo r s i gn i f i can t an t i f unga l act iv i ty h o w e v e r no m i n i m a l inh ib i tory c o n c e n t r a t i o n s w e r e r e p o r t e d . It is i n te re s t i ng to n o t e in t h e s e t w o p u b l i c a t i o n s the e f fec t of the r e m o v a l of 2 ' - h yd roxy l a s i l l u s t ra ted in F i g u r e 4.11 a n d 4.12. It w o u l d s e e m that 2 ' h yd r ox y l g r o u p p l a y s a s i gn i f i c an t ro le in d e c r e a s i n g an t i f unga l act i v i ty p r o b a b l y d u e to its w i t h d r a w i n g e l e c t r o n i c e f fect . Active Inactive Figure 4.13 Effect on Antifungal Activity of the Removal of 2'-Hydroxyl (Sato, 1994) P i n o c e m b r i n h a s b e e n r e p o r t e d in lea f r e s i n of e a s t e r n c o t t o n w o o d (Populus deltoides) ( S h a i n a n d Mi l le r , 1982 ) , a e r i a l pa r t s o f Teloxys graveolens ( C a m a c h o et al., 1991 ) , in h e a r t w o o d o f Pseudotsuga menziesii (Doug la s - f i r ) (De l l u s et al., 1997 ) , a e r i a l pa r t s a n d roo t s of Lippia graveolens ( O r e g a n o ) ( D o m i n g u e z , et al., 1989) ; a n d a l o n g w i th c h r y s i n in l e a v e s o f Anomianthus dulcis 119 (Sinz et al., 1999). Galangin, chrysin and pinocembrin, among others, are characteristic flavonoids from propolis (bee glue) and bee wax. However no studies have been published in regards to ecological role for pinocembrin or its chalcone. The large amount of taboganic acid extracted from the plant (3.06 g) is noteworthy. Although no activity was shown against C. albicans, this compound presented activity against the plant pathogenic fungus Cladosporium cucumerinum in a bioautographic test on TLC (5 pg), although it did not exhibit activity in a dilution assay (Terreaux et al., 1998). Its methyl ester showed activity in the leaf cutter repellence assay and consequently it might constitute a natural defence against ants (Roussis etal., 1990). In regards to antifungal activity of chalcones and their structure-activity relationships, S . N. Lopez and co-workers (2001) tested a series of chalcone derivatives (this list does not include pinocembrin chalcone) against known dermatophytes (Mycrosporum canis, M. gypseum, Trichopyton rubrum and Epidermophyton floccosum). The authors' conclusions were that planar forms in chalcones have the spatial ordering needed to produce the antifungal response, whereas p- and carbonyl carbons are most susceptible sites for a possible nucleophilic attack of the enzyme or receptor. However none of the compounds tested were active against Candida albicans, Saccharomyces cerevisae or Cryptococcus neoformans nor against the filamentous fungi Aspergillus niger, A. fumigatus or A. flavus. 120 It h a s b e e n r epo r t ed that a s l ight s t ruc tu ra l m o d i f i c a t i o n o f p i n o c e m b r i n c h a l c o n e b r o u g h t a n i n c r e a s e in act iv i ty a g a i n s t S. aureus M S . I ndeed , 2 ' , 6', 4 -t r i h yd roxy - 4 - m e t h o x y d i h y d r o c h a l c o n e ( a s e b o g e n i n ) e x h i b i t e d act iv i ty a g a i n s t S. aureus M S , a l t h o u g h the m i n i m a l inh ib i tory c o n c e n t r a t i o n is not r epo r t ed ( J o s h i et al., 2001) . \ Figure 4.14 Asebogenin (2', 6', 4 - trihydroxy - 4 - methoxydihydrochalcone) (Joshi, 2001). R e g a r d i n g t he s e a r c h f o r n e w inh ib i to r s f o r meth i c i l l i n - r e s i s t an t Staphylococcus aureus s t r a in s , c h a l c o n e s s e e m to d i s p l a y h i g h e r act iv i ty t h a n f l a v o n e s a n d f l a v a n o n e s T h e c a r b o n y l i c r e g i o n a l o n g w i th m o l e c u l a r c o - p l ana r i t y m a y p l ay a n impo r t an t ro le in a n t i - M R S A act i v i t y ( A l c a r a z , et al., 2000). 121 4.5. CONCLUSION It i s c r u c i a l to b e a r in m i n d that t he t rad i t i ona l u s e o f th i s p l an t i s a n a q u e o u s e x t r a c t u s e d ex te rna l l y to a l l e v i a t e s k i n c ond i t i o n s . T h e two c o m p o u n d s f o u n d to b e a c t i v e p r e s e n t e d a w e a k act i v i ty a g a i n s t f ung i a n d b a c t e r i a . M i n i m a l inh ib i to ry c o n c e n t r a t i o n s s u c h a s t he o n e s r e p o r t e d h e r e c a n n o t s e n s i b l y b e r e g a r d e d a s m e a n i n g f u l . A l s o , no s y n e r g y e f f e c t s w e r e o b s e r v e d fo r the i s o l a ted c o m p o u n d s . O n t he o t h e r h a n d , t he f ac t that t h e a q u e o u s e x t r a c t d i d no t p r e s e n t act iv i ty a g a i n s t C. albicans or T. mentagrophytes c h a l l e n g e s o u r h y p o t h e s i s e v e n fu r ther b e c a u s e th i s is t he ex t rac t l i ke ly to b e o b t a i n e d by t h e t rad i t i ona l m e t h o d . I a m t e m p t e d to a f f i rm that t h e t rad i t i ona l u s e o f P. lanceaefolium in t h e S i b u n d o y v a l l e y h a s no th i ng to d o wi th c o u n t e r a c t i n g i n f e c t i ou s a g e n t s s u c h a s b a c t e r i a , fung i o r v i r u s e s . It i s p o s s i b l e that t he t rad i t i ona l t r e a t m e n t i s m e a n t t o p h y s i c a l l y and/or t o p s y c h o l o g i c a l l y a l l e v i a t e t he s y m p t o m s o f d i s c o m f o r t c a u s e d by a s t i l l - to -be -ident i f ied a gen t . A s s o c i a t i o n w i th t he m o o n m a y s i gn i f y t he c y c l i c a p p e a r a n c e of s u c h a n a gen t . 122 4.6. References Alcaraz, L.E.; Blanco, S.E. ; Puig, O.N.; Tomas, F.; Ferretti, F.H., 2000. Antibacterial activity of flavonoids against Methicillin- resistant Staphylococcus aureus strains. Journal of Theoretical Biology 205: 231-240. Ampofo, S.A., Roussis, V.^and Wiemer, D.F. 1987. New Prenylated Phenolics from Piper auritum. Phytochemistry 26: 2367-2370. Bremner, P.D. and Marion Meyer, J . J . 1998. Pinocembrine Chalcone: An Antibacterial Compound from Helichrysum trilineatum. Planta Medica 64: 777. Camacho, M. R.; Sanchez, B.; Quiroz, H.; Contreras, J.L. and Mata, R. 1991. Pinocembrin: a Bioactive Flavanone from Teloxys graveolens. Journal of Ethnopharmacology 31: 383-389. Dellus, V.; Mila, I.; Scalbert, A.; Menard, C ; Michon, V.; Herve du Penhoat, C .L .M. 1997. Douglas-fir Polyphenols and Heartwood Formation. Phytochemistry 45: 1573-1578. i Diaz, D.P.P.; Tiberio, A .C . and Joseph-Nathan, P. 1987. A Chromene, an isoprenylated methyl hydroxybenzoate and a C-methyl flavanone from the bark of Piper hostmannianum. Phytochemistry 26: 809-811. 123 Dominguez, X.A.; Sanchez, H.; Suarez, M.; Baldas, J . H.; and Gonzalez, M.R. 1989. Chemical Constituents of Lippia graveolens. Planta Medica 2: 208-209. Joshi, A. S. ; Li, X . - C ; Nimrod, A. C ; ElSohly, H. N.; Walker, L. A. and Clark, A. M. 2001. Dihydrochalcones from Piper longicaudatum. Planta Medica 67: 186-188. \ Fukui, H.; Goto, K.; Tabata, M. Two Antimicrobial Flavanones from the Leaves of Glycyrrhiza glabra. 1988 Chemical Pharmaceutical Bulletin 36: 4174-4176. Lopez, S .N. ; Castelli, M.V.; Zacchino, S.A.; Dominguez, J .N . ; Lobo, G. ; Charris-Charris, J . ; Cortes, J . C . G . ; Ribas, J . C ; Devia, C ; Rodriguez, A . M . and Enriz, R.D. 2001. In Vitro Antifungal Evaluation and Structure-Activity Relationships of a New Series of Chalcone Derivatives and Synthetic Analogues, with Inhibitory Properties Against Polymers of the Fungal Cell Wall. Bioorganic and Medicinal Chemistry 9:1999-2013. Mc Dowell, J H . 1989. Sayings of the Ancestors. The spiritual life of the Sibundoy indians. The University Press of Kentucky. Lexington, p.77. i Mundina, M.; Vila, R.; Tomi, F.; Tomas, X.; Ciccio, J .F. ; Adzet, T.; Casanova, J . and Canigueral, P. 2001. Composition and chemical polymorphism of the essential oils from Piper lanceaefolium. Biochemical Systematics and Ecology 29: 739-748. 124 Parmar, V. S.; Jain, S. C ; Bisht, K. S.; Jain, R.; Taneja, P.; Jha, ; A., Tyagi, O. D., Prasad, A. K.; Wengel, J . ; Olsen, C. E.; Boll, P. M. 1997. Phytochemistry of the Genus Piper. Phytochemistry 46:597-673. Roussis, V.; Ampofo, S.A. and Wiemer, D.F. 1990. A Prenylated Benzoic Acid Derivative from the Leaves of Piper taboganum. Phytochemistry 29: 1787-1788. Rahalison, L., Hamburger, M.; Monod, M.; Frenk, E.; Hostettmann, K. 1994. Antifungal tests in phytochemical investigations-comparison of bioautographic methods using phytopathogenic and human pathogenic fungi. Planta Medica 6 0 : 41-44. Saxena, G.; Farmer, S.; Towers, G. H. N.; Hancock, R. E. W. 1995. Use of specific dyes in the detection of antimicrobial compounds from crude plant-extracts using a thin-layer chromatography agar overlay technique. Phytochemical Analysis 6 : 125-129. Sato, M.; Tsuchiya, H.; Akagiri, M.; Fujiwara, S. ; Fujii, T.; Takagi, N.; Matsuura, N. and linuma, M. 1994. Growth Inhibitory Properties of Chalcones to Candida. Letters in Applied Microbiology 18: 53-55. 125 S c h u l t e s , R. E. a n d Raf fau f , R. F. 1992 . T h e H e a l i n g F o r e s t : M e d i c i n a l a n d T o x i c P l a n t s of the N o r t h w e s t A m a z o n i a . Edited by: T h e o d o r e R D u d l e y . D i o s c o r i d e s P r e s s . P o r t l a n d , p 3 6 4 . S h a i n , L. a n d Mi l le r , J . B . 1982 . P i n o c e m b r i n : a n A n t i f u n g a l C o m p o u n d S e c r e t e d by L e a f G l a n d s of E a s t e r n C o t t o n w o o d . Phytopathology 72:877. S i n z , A.; M a t u s c h , R.; E l s a c k e r , F.; S a n t i s u k , T.; C h a i c h a n a , S. a n d R e u t r a k u l , V . 1999 . P h e n o l i c C o m p o u n d s f r o m Anomianthus dulcis. Phytochemistry 50: 1 0 6 9 -1072 . Tay l o r , R.S.L., M a n a n d h a r N.P., T o w e r s G . H . N . 1 9 9 5 . S c r e e n i n g of s e l e c t e d m e d i c i n a l - p l a n t s f o r N e p a l f o r a n t i m i c r o b i a l a c t i v i t i e s , dournal of Ethnopharmacology 46: 1 53 - 159 . T e r r e a u x , C ; G u p t a , M . P.; H o s t e t t m a n n , K. 1 998 . A n t i f u n g a l B e n z o i c A c i d D e r i v a t i v e s f r o m Piper dilatum. Phytochemistry 49: 4 6 1 - 4 6 4 . T r e l e a s e , W . 1950 , T h e P i p e r a c e a e o f N o r t h e r n S o u t h A m e r i c a . U n i v e r s i t y o f i I l l inois P r e s s . U r b a n a . pp . 193 -194 . T s u c h i y a , H.; S a t o , M.; A k a g i r i , M. ; T a k a g i , N.; T a n a k a , T.; l i n u m a , M . 1994 A n t i -Candida A c t i v i t y o f S y n t h e t i c H y d r o x y c h a l c o n e s . Pharmazie 49: 7 5 6 - 7 5 8 . 126 Warrier, P. K.; Nambiar, V. P. K.; Ramankutty, C. 1995. Indian Medicinal Plants. Vol. 4. Orient Longman. Madras, pp. 279-303. Zanchin, G. 2001. Macro and Microcosmus: Moon Influence on the Human Body. Earth Moon and Planets 85-86: 453-461. 127 Chapter 5 Biological Activities from Iryanthera megistophylla A.C. Sm. Compounds Find a toad generous in size, And wash its belly thoroughly. Hold the toad by its limbs and gently Rub the stomach on the affected area. Crucify the toad at dawn and cremate after death. Repeat the treatment for eight days. Home remedy for Herpes 5.1. Introduction Iryanthera is a neotropical genus and some of its species are used in the treatment of different ailments. The Waorani from Ecuador for instance, shred the inner bark of /. juruensis , I. paraensis or /. elliptica and use the "resin" to treat fungal infections of the skin. They apply the resin directly onto infected areas, where it kills the fungus "just like the dart poison" (Davis and Yost, 1983). Likewise the Puinaves from Colombia employ the soft inner bark of Iryanthera species on areas of fungal infection of the skin (Schultes and Raffauf, 1990). On the other hand /. tessmannii is used in The Iquitos (Peru) region for treatment of diarrhoeas. In Peru the resin of the bark of /. ulei is used in the treatment of "patco", a disease in which a white substance appears in a child's mouth (Schultes and Raffauf, 1990). The reference unfortunately does not provide further information about this ailment. Whereas Iryanthera extracts' are 128 used as anti-fungal agents in the Amazon Basin, the Afro-Colombian communities of the Pacific Coast use it to treat cutaneous leishmaniasis. The phyto-chemistry of this genus has been studied (Vieira et al., 1983, Garzon et al., 1987, Conserva etal., 1990, Silva etal., 2001). The distribution of flavonoids has been reviewed (Martinez, 2000) and Ming and co-workers (2002) have studied the phyto-chemistry of Iryanthera megistophylla. Based on the results of the anti-viral and anti-bacterial assays described in chapter 3, Iryanthera megistophylla appeared to be a good candidate to undertake phytochemical analysis. Indeed not only did the methanolic extract of this plant display good activity against herpes simplex virus but also against Staphylococcus aureus methicillin-sensitive M S . This extract presented good inhibition against secreted aspartic proteases in the inhibition test described chapter 3 (Section 3.2.6.2.2). In this chapter I will describe and discuss the anti-viral and anti-microbial activity of four of the seven compounds isolated by Dr. Dong Sheng Ming from /. megistophylla. Also I will describe and discuss the inhibitory activity of these compounds against secreted aspartic proteases from C. albicans. Considering the chemical nature of some of the isolated compounds (flavolignans) and their putative role in inhibiting resistance pumps, I will describe and discuss the multidrug resistance test (Guz et al., 2001). The activity of iryantherin K against a battery of Gram positive, Gram negative and yeast will be presented along with its cytotoxicity in a mammalian 129 system. The potential of iryantherin K in the treatment of Staphylococcus aureus methicillin-resistant MR infections will be discussed. 5.2. Materials and Methods 5.2.1. Anti-viral assays. Determination of Minimal Inhibitory Concentration (MIC) \ Assays were carried out with 100 plaque-forming units (PFU) of HSV-1 in Vero cells grown in 96-well culture trays, as described in chapter 3 section 3.2.3. The MIC is the minimum concentration of compound, which caused complete inactivation of virus infectivity (i.e., absence of viral C P E ) . A methanolic leaf extract of Adansonia digitata was used as a positive control (minimum antiviral concentration in UVA, 5 pg/mL) (Hudson et al., 2000). 5.2.2. Cytotoxicity Assay 5.2.2.1. Cytotoxicity Visible Assessment To test for cytotoxicity, Vero cell monolayers were grown in 96-well microtiter plates (Falcon) and exposed to serial dilutions of the corresponding compounds starting at 320 pg/mL. The treated cells were then incubated at 37°C for 1 h, exposed to UV-A light and visible light for 30 min and then re-incubated i for 24 hrs. The cells were examined periodically through the microscope for assessment of changes in cell morphology or visible toxic effects (obvious cellular damage or lysis). 130 5.2.2.2. Cell Proliferation Assay for Iryantherin K. M o u s e f i b rob l a s t c e l l s ( N I H 3 T 3 ) w e r e s e e d e d at 1 0 0 0 ce l l s /we l l in 9 6 - w e l l p l a te s , g r o w n ove rn i gh t , a n d t r e a t ed or not t r ea ted w i th i r y an the r i n K in a d i lu t ion s e r i e s (50-0.001 pg/mL) for 2 4 hou r s . I ryanther in K w a s r e m o v e d , a n d c e l l s w e r e a l l o w e d to g r o w in f r e s h m e d i u m unti l t h o s e not t r e a t ed w i t h t he c o m p o u n d a p p r o a c h e d c o n f l u e n c e , w h i c h w a s t yp i ca l l y 4 -6 d a y s . C e l l p ro l i f e ra t i on w a s m e a s u r e d a s f o l l ows : 2 5 p L o f a 5 mg/mL s o l u t i on o f 3 ( 4 , 5 - d ime thy l t h i a zo l - 2 - y l ) -2 , 5 - d i p h e n y l t e t r a z o l i u m b r o m i d e in p h o s p h a t e - b u f f e r e d s a l i n e w a s a d d e d to ce l l s in the p r e s e n c e o f 100 p L of c e l l c u l t u r e m e d i u m . A f t e r a 2 h o u r s i n c u b a t i o n at 3 7 °C, 1 00 p L of 2 0 % s o d i u m d o d e c y l s u l f a t e d i s s o l v e d in d i m e t h y l f o r m a m i d e / w a t e r (1:1), p H 4.7, w a s a d d e d , a n d t he a b s o r b a n c e at 5 7 0 n m w a s m e a s u r e d af ter o v e r n i g h t i n c u b a t i o n ( C u r m a n et al., 2 0 0 1 ) . 5.2.3. Antibacterial and Antifungal Assays. 5.2.3.1 Minimum Inhibitory Concentration (MIC) determination A s d e s c r i b e d in 4.2.6 5.2.3.2 Test for Multidrug Resistance (MDR) Inhibitory Activity S. aureus A T C C 2 5 9 2 3 M S (meth i c i l l i n - s en s i t i v e ) w a s c u l t u r e d in 3 m L o f M u e l l e r - H i n t o n ( M H ) broth o ve r n i g h t at 3 7 ° C . C e l l s w e r e t h e n d i l u ted 1:2000 into M H broth ( 4 . 1 x 1 0 8 c e l l s /mL ) a n d 0.05 m L w e r e d i s p e n s e d into e a c h w e l l of m ic ro t i te r p l a te s . T e s t s u b s t a n c e s w e r e se r i a l l y d i l u ted 2 - fo ld in the w e l l s for a v o l u m e o f 0 . 075 m L p e r w e l l . A m e t h a n o l i c s o l u t i o n o f b e r b e r i n e (0 .075 m L ) w a s a d d e d to e a c h w e l l at a s ub - i nh i b i t o r y c o n c e n t r a t i o n (30 pg/mL, 1/8 M IC ) . M i n i m a l 131 inhibitory concentration was defined as the minimal concentration that completely inhibited cell growth in presence of 30 pg/mL of berberine. The final volume of the well was 0.2 mL. (Guz ef al., 2001). 5.2.4. Secreted Aspartic Proteases Inhibition Assay Secreted Aspartic Proteases Inhibition Assay for isolated compounds is de in section 4.2.8. IC 50 values were derived from the dose-effect curves calculated according to the regression analysis (least-square method) by plotting the log concentration against the percentage of inhibition. 5.2.5. Pepsin Inhibition Assay Optimum final pepsin (from Porcine Stomach Mucosa- Sigma) and substrate concentrations were determined to be 15 U/mL (471 Units/mg enzyme) and 40 pM respectively. The assay was performed as for secreted aspartic proteases except pepsin was substituted for the S A P extract and 50 mM sodium citrate buffer (pH= 4.0) was used as diluent. 5.3. Results 5.3.1 Cytotoxicity i None of the compounds (1-9) presented visible cytotoxic effects were at concentrations up to 320 ug/mL. In the cell proliferation assay iryantherin K was found to be cytotoxic at 50 pg/mL but it did not present cytotoxic effects at lower concentrations (10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005 and 0.001 ug/mL). 132 5.3.2 Antiviral and Antimicrobial activities B i o l o g i c a l ac t i v i t i e s of i s o l a t ed c o m p o u n d s a r e s u m m a r i s e d in T a b l e s 5.1; 5.2 a n d 5.3. C o m p o u n d s 6 a n d 7 ( S e e F i g u r e s 5.1 a n d 5.2) w e r e f o u n d to b e a c t i v e a g a i n s t H S V - 1 at a m i n i m a l inh ib i tory act iv i ty of 2 0 pg/mL. Figure 5.1 Structure of Cinchonain lb (6) Figure 5.2 Structure of Cinchonain la (7) 133 Figure 5.3 Procyanidin B 2 or catechin - (4p - 8) epicatechin (8) Figure 5.4 Structure of Cinchonain Ha (9) 134 LO 3 > 5 0) o o_ E o u •2 •c a o o CO c o ro i_ +J c a> u c o o o c "ro E 'E « to CO 0) a E W T (A .E a> = 8- o * !E ro o£ I? * co T3 C 3 2! ro CO "D C ro > 0) c o !E cu E. 3 2 3 ro CO </> c ro o ro 6 S. aureus MR SAP 0017 (clinical isolate) Q Q Q L O Q Q Q Q Q Z Z Z C M Z Z Z Z Z S. aureus MS (Mg/mL) ATCC 25923 MDR Test * \ • \ LO LO o O C N C N o O S. aureus MS (Mg/mL) ATCC 25923 o o o i£ <-> o O o I I I 2 2 I I ° I albicans 'Mg/mL) ^ n .o .o .o O O O O I I I I I £ £ £ £ d HSV-1 (MQ/mL) to ro ro ro ro Q Q ro ro I I I I I C N C N I I spunod Com| CO CD _ , h O) O) E E •o CD 0) "CD T 3 O C CO CO i s <Z CO CD £ : 3 . c O Ig x: _c CD O c t o ' co CD O o CN O co oo CO CO > •> O o CM 4—» co > CD o c o o o o CO _CD X) ro CD O =3 CD - « t-i '*= -Q o CO 0) X 2 CO •4—• o £ "CD "O o c CO E "c E Q Z T3 CD > O CD CL CO CD fL -00 co CO CO T3 C CO CO c CO o co d _to o c o o CD .> •4—' CO o CL CO CO -Q CD CO CD i— CD 'o E CO •4—' rz CD CD T3 C CO c o "i_ CD o CL E < 135 .2 *-> u TO CQ 0 > "43 "55 o Q. E ra L . o (/> c "ro co ro * c Q) CM ra in *> Z X2 O ra ^ CO CO c o "43 ra 4-1 C Q) O c o CJ o ra E "E B.subtilis ATCC 6633 wt LO 0 0 £. faecalis ATCC 25912 wt ^ £ CD CO S.epidermidis C621 wt LO ^ CD CM CNJ — r - CO S.aureus ATCC 25923 wt £ CM £ ™ -r~ d S. aureus SAP0017 MR 2.5 16 (64) 16(32) 32 Compounds Iryantherin K Methicillin Polymyxin B Gentamicin CD CO c ro CO CD o _ro TD o a* CM S rol £ ° CD CD J= ro ro _ £ C N CO C D o w .CO "35 !Q s 1 .£ & « ro i l £ ro ° w CD ^1" CM c CD = ro : y c £ o E w <2 CO 3 P Z> 3 CO TO CD a: co re CD O re CQ a > "43 re co a> c E re L -(D co c "re CO re c . c ^ ro 0 — 1* CO (0 c o re i_ •*•> c CD o c o o o .JZ _c "re E "E S.typhimurium C610 abs >20 >128 0.125 S.typhimurium ATCC 14028 wt „ oo ° ^ - s E.coli DC2 abs O CO LO CM ^ q CN A O O E.coli UB1005 wt >20 >128 0.125 0.125 P.aeruginosa H188 abs >20 0.5 (2) 0.125 0.125 P.aeruginosa H187 wt >20 128 0.125 0.125 Compounds Iryantherin K Methicillin Polymyxin B Gentamicin CO c 'ro co oo CD TO fN CD £ C CD J Z o T J o 4— CN c ro o o jn y> o o o o c ro X CD ro < E 3 CD Q 3 .E •£ CM ^ CO 4—* CD o CD i— n c c o .c CO CO T3 c ro io o o m ID O o £ LU oo co" Z J O CN CD ro c $ o SZ CO CO oo 0 0 "D c ro oo CO o c ro ZJ E CD > CO c CD CO cz 'co c a CD •o ro CO C O _ O 8 J §>.§ 5 * CD §-CD -CT Z J CO CD a: a. co 136 Iryantherin K (4) and iryantherin L (5) are active against S. aureus MS (methicillin-sensitive) at 1.25 pg/mL and iryantherin K (4) presented very good activity (2.5 pg/mL) against S. aureus MR (methicillin-resistant). Also it presented very good activity against others Gram positive bacteria (see Table 5.2). There was inadequate material to test other compounds. Compounds 6 and 7 showed a weak activity in the multidrug resistance inhibitory test. Figure 5.5 Iryantherin K (4) 137 Figure 5.6 Iryantherin L (5) The MIC for compounds 6,7,8,9 against C. albicans was of 150 pg/mL The remaining compounds were not active against C. albicans or S. aureus methicillin-sensitive MS at the upper concentration tested in the broth dilution assay (300 and 200 pg/mL, respectively) (Table 5.1). Compounds 1, 2 and 3 were not active in any of the tested systems. 138 Figure 5.7 Megislignan (1) [2,3-dimethyl-4-(4-methoxyphenyl)-6-hydroxynaphthalene] 16' 14' 1' O H Figure 5.8 Megislactone (2) [(2R,3R,4R)-3-hydroxy-4-methyl-2-(hexacos-17-enyl) butanolide] 139 5.3.3. Secreted Aspartic Proteases Inhibition Only compounds megislignan (1), iryantherin K (4) and iryantherin L (5) were inhibitory at 40 pM in the secreted aspartic protease inhibition assay. Table 5.4 Secreted Aspartic Proteases Inhibition Activity of compounds from /. megistophylla Compound Inhibitory activity (%) 1 12 4 64 5 42 Compounds No 2, 3, 6,7, 8 and 9 did not present activity 140 5.3.4. Inhibitory Activity of Iryantherin K (4) and L (5). Iryantherin K and L were selected for evaluation of effects against Candida albicans secreted aspartic proteases. The IC50 values were calculated based on the corresponding log-dose curve, (See Table 5.3). Pepstatin, which was used as a positive control, showed an IC50 of 0.003 pg/mL. To determine if the inhibitory effects of these compounds were selective for Candida secreted aspartic proteases (SAP), their activities against an aspartic protease, pepsin, were determined. Due to limitations in the amount of material the inhibitory activity against pepsin was determined at a concentration close to the compounds' IC50 values. Iryantherin K caused inhibition of 56.2% at 15 pg/mL while Iryantherin L presented a value of 37.6% at 20 pg/mL. 141 <A C ro u re CO Q. < <A (4) (A (0 <D O ro a (A < n LO <D 0) (0 •g « " I (0 CO ra • o c re CD x : c re £ o > o < o 15 IE c c c o 'to £ 3 a> • - —. a. -E o LO o o O E "3) c o re k_ •*-> c a> u c o O CN E LO 5 2 E o oo LO CN o CN LO LO T3 C 3 O a E o O CD oo CN CD LO 0 0 CD CO LO O i o CN CN -H co oo o -H c\i oo co d -H CD LO -H CN CO LO d -H OO CN CD d -H . co c a) sz ' c co o CN CO CN 0 0 LO d -H LO T f CN 0 0 d -H CN LO CN CO d -H CO 0 0 co CO CN LO 0 0 d -H d o +i CO CO x : c co 142 5.4. Discussion Cinchonain lb (6) and la (7) (See Figure 5.1 and 5.2) showed potent activity against HSV-1 at a minimal concentration of 20 ug/mL (Table 5.1). Compounds 6 and 7 were thus partly responsible for the antiviral activity detected in the crude extract of /. megistophylla (Lopez et al., 2001). The fact that the crude extract exhibited a minimal inhibitory concentration of 10 ng/mL (See Table 3.1 in Chapter 3) and 20 ^ g/mL for each one of the pure compounds, might indicate that a synergistic effect took place which was responsible for the overall observed anti-viral effect. Cinchonain lb (6) and la (7) were first isolated by Nonaka and co-workers (1982) from the bark of Cinchona succirubra, which is the source of quinine and quinidine. They have been isolated as well from Uncaria rhynchophylla (Rubiaceae), Kandelia candel (Rhizophoraceae), Polygonum bistorta (Polygonaceae) and Raphiolepsis umbellata (Rosaceae). They exhibited poor activity (150 pg/mL) against C. albicans and were inactive against S. aureus MS. Aside from the activities reported here, compound 7 has also been shown to be hepatoprotective. It has been suggested that this compound, by scavenging reactive oxygen species (ROI's), interferes with the signal transduction triggered by TNF-a (activator of phagocytic cells) and thus protects the cell from subsequent injury (Fan et al., 1999; Xiong et al., 2000). One can speculate that because of the polyphenolic nature of compounds 6 and 7, they are likely to bind to the protein coat of the virus or to the host's cell membrane. It is believed that virus adsorption and consequently virus penetration 143 is t hu s a r r e s t e d . F o r i n s t a n c e , F u k u c h i ef al ( 1989 ) u s i n g r a d i o l a b e l e d v i ru s pa r t i c l e s s h o w e d the ant i -v i ra l ac t i v i t ie s o n H S V - 1 a n d H S V - 2 of h i gh m o l e c u l a r w e i g h t p o l y p h e n o l s (i. e. t a n n i c a c i d , co r i a r i i n A , r u g o s i n D ) o n A f r i c a n g r e e n m o n k e y k i d n e y c e l l s w e r e d u e to the inh ib i t ion of v i r u s a d s o r p t i o n . In th is r e g a r d , p r o c y a n i d i n B-2 (8) ( F i g . 5.3) a n d c i n c h o n a i n I la (9) ( F i g . 5.4), w h i c h a r e c h a r a c t e r i s e d by a l a r ge n u m b e r o f p h e n o l i c g r o u p s , d i d not exh ib i t an t i v i r a l act iv i ty to H S V - 1 in th is a s s a y . In a n e x t r a c e l l u l a r v i r uc i da l ( ce l l s p r e - t r ea ted w i th v i rus ) a s s a y it w a s f o u n d that p r o c y a n i d i n B-2 r e d u c e d the n u m b e r o f p l a q u e s to 5 0 % at 3 5 pg/mL ( T a k e c h i et al., 1985 ) . A l s o p r o c y a n i d i n B-2 at 100 pg/mL p r e s e n t e d 1 0 4 fo ld r e d u c t i o n of H S V - 1 v i r u s t iter in V e r o c e l l s ( De B r u y n e , etal., 1999a ) . P r o c y a n i d i n B-2 o r c a t e c h i n - ( 4 0 - 8) e p i c a t e c h i n (8) i s a w e l l - k n o w n p r o a n t h o c y a n i d i n ( p rev i ou s l y re fe r red to a s " c o n d e n s e d " t ann i n s ) , w h i c h a r e o l i g o m e r s a n d p o l y m e r s c o m p o s e d o f f l a v a n - 3 - o l n u c l e i ( D e B r u y n e e f al., 1999b ) . It h a s b e e n r e p o r t e d to h a v e h y d r o x y l r a d i c a l s c a v e n g i n g act iv i ty a n d p r e s e n t e d inh ib i to ry act iv i ty in a m i c r o s o m a l l ipid p e r o x i d a t i o n a s s a y (ICso= 3.3 J I M ) ( S h a h a t et al., 2 002 ) . P r o c y a n i d i n B-2 (8) a n d c i n c h o n a i n lb (6) e xh i b i t ed a n t i l e i s h m a n i a i ac t i v i t y in vitro ( K o l o d z i e j et al., 2 0 0 1 ) , w h i c h m a y e v e n t u a l l y e x p l a i n the t rad i t i ona l u s e of /. megistophylla in the t r e a t m e n t o f l e i s h m a n i a s i s . P r o c y a n i d i n B-2 s h o w e d w e a k ac t i v i t y a g a i n s t S. aureus M S (M IC=100 pg/mL) a n d a g a i n s t C. albicans ( M I C = 1 5 0 pg/mL). T h e s e c o n c e n t r a t i o n s c a n n o t b e r e g a r d e d a s m e a n i n g f u l b e c a u s e of t he h igh c o n c e n t r a t i o n o f c o m p o u n d that h a s to b e u s e d to inhibit t he act iv i ty. T h e c o n c e n t r a t i o n o f th i s c o m p o u n d in the 144 ethanolic extract was very low compared to the amount found for iryantherin K for example (for iryantherin K 0.4% referred to the ethanolic extract). There is indeed a tendency to allocate biological activities of tannins because of their capacity to bind proteins in a non-specific manner. However, on the one hand, characteristics of both the tannin and the protein as well as the conditions of reaction influence tannin-protein interactions. On the other hand it has been demonstrated that some phenolic compounds (i.e. procyanidin B-2) show specific activities at receptor levels which cannot solely be explained in terms of non specific protein binding (Zhu et al., 1997). Iryantherin K (4) and L (5) have been isolated previously from bark of /. ulei (Conserva et al., 1990) and the pericarp of /. lancifolia (Silva et al., 1999). These iryantherins belong to a complex group of dihydrochalcones that have been isolated exclusively from Iryanthera species (so far). Iryantherin K and its stereoisomer iryantherin L showed good activity against S. aureus MS (MIC=1.25 pg/mL). Also iryantherin K was the more abundant compound in the ethanolic extract whereas iryantherin L was present only in small amounts. More importantly, iryantherin K presented potent activity against S. aureus MR (methicillin-resistant) and presented no cytotoxic effects at 10 pg/mL in mouse fibroblast cells. This may prove a very important finding from the therapeutic perspective. .Iryantherin K and L had inhibitory effects against C. albicans secreted aspartic proteases. These compounds were also active in the same range of concentrations, against the aspartic protease pepsin. It is not surprising since the 145 overall architecture of the SAP ' s from Candida conforms with the classical aspartic protease fold typified by pepsin (Sielecki et al., 1990). However, the structures of the SAP ' s from Candida present unique features in both the amino and carboxy lobes (Abad-Zapatero, 1998). Considering that secreted aspartic proteases are a virulence factor in candidosis it would be interesting to test these compounds in an animal model. Interestingly, compounds 4 and 5 have been found to have potent antioxidant activities as measured by their ability to inhibit spontaneous lipid peroxidation of brain homogenates (Silva et al., 1999). A necessity to maintain the chemical integrity of fruit tissue rich in easily oxidizable fatty acids and triglycerides has been suggested (Silva ef al., 1999). In regards to antioxidant activity it has been suggested that flavonoids multiple biological activities may result, at least, in part from their antioxidant and free radical-cavenging abilities. The protective effect of flavonoids against membrane lipoperoxidative damage has been well established, and seems to depend both on their structure and ability to interact with and penetrate the lipid bilayers. During pathological processes reactive oxygen species accumulate and the mitochondria membrane may undergo lipid peroxidation and/or increase in permeability. Agents that inhibit these processes may be of high pharmacological potential. However it has been shown that same flavonoids could behave as both antioxidants and prooxidants, depending on concentration and free radical source (Cao ef al., 1997). 146 Although members of the genus Iryanthera are traditionally used to treat fungal infections, no significant activity was found against the yeast C. albicans (MIC=150 ug/mL). It may be worth testing these compounds against other fungi, such as dermatophytes, that are found in condit ions simi lar to the site of col lect ion. It is a lso important to recal l the fact that it is the resin that is used to treat fungal infections rattier than the bark. In general resins are composed mainly of terpenoid- like compounds that have been shown to have a wide range of antibiotic activity. However very few aspart ic proteases have been isolated up to now in bacter ia, and those descr ibed so far s e e m not to be involved in pathogenesis (Supuran ef al., 2002). C inchona in lb (6) and la (7) presented weak synergist ic activity in the p resence of berberine against S . aureus M S at minimum inhibitory concentrat ion of 100 pg/mL. In the absence of berberine these compounds do not present antibiotic activity. A number of f lavol ignans have been found to act as potentiators of otherwise weak ant imicrobials and it has been suggested that their act ion is due to inhibition of the S . aureus M D R efflux pump protein NorA (Guz et al., 2001). It would be interesting to compare these S . aureus NorA putative inhibitors with some inhibitors of the mammal ian M D R P-gp efflux proteins s ince any inhibitor of a microbial M D R pump deve loped for therapeutic i use should not affect P-gp, which plays a role in xenobiot ic efflux of normal t issues. 147 Although c inchonain lb and la presented synergist ic activity in the p resence of berberine, they did not potentiate any activity in the p resence of iryantherin K (4) nor iryantherin L (5). This indicates that there is no antimicrobial synergist ic effect amongs t the compounds found in this plant. 5.5. Conclusion The anti-viral activity of Iryanthera megistophylla reported in Chapter 3 might be due to a synergist ic effect amongst different compounds . In a b io-assay guided phytochemical separat ion, c inchonain lb (6) and la (7) were found to have anti-viral activity against herpes s implex virus. It is poss ib le that the polyphenol ic nature of these compounds is related with the anti-viral activity but no definitive ev idence has been brought forward. A l so it would be worth investigating an eventual correlat ion between inhibition of aspart ic proteases and the anti-oxidant propert ies reported e lsewhere. Iryantherin K activity against S . aureus M R represents an important finding and it might hold promise as a new powerful antibiotic act ive against this resistant bacter ia. 1 4 8 5.6. References Abad-Zapatero, C . 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Proanthocyanid ins and Re la ted C o m p o u n d s : Ant i le ishmania l Activity and Modulatory Effects on Nitric Ox ide and Tumor Necros is Fac to r -a -Re lease in the Murine Macrophage-L ike Ce l l Line R A W 264.7. Biological and Pharmaceutical Bulletin 24: 1016-1021. Lopez , A . ; Hudson , J . B.; Towers , G . H. N. 2 0 0 1 . Antiviral and antimicrobial activities of Co lomb ian medicinal plants, dournal of Ethnopharmacology 77: 189-196. i Mart inez, J . C . 2000. Distribution of f lavonoids in the Myr is t icaceae. Phytochemistry 55: 505-511. 151 Ming, D.S., Lopez, A., Hillhouse, B.J. , French, C . J . , Hudson, J .B. and Towers, G.H.N. 2002. Bioactive Constituents from Iryanthera megistophylla. Journal of Natural Products 65: 1412-1416. Nonaka, G-l and Nishioka, I. 1982. Tannins and Related Compounds. VII. Phenylpropanoid-substituted Epicatechins, Cinchonains from Cinchona succirubra. Chemical and Pharmaceutical Bulletin 3 0 : 4268-4276. Schultes, R.E. and Raffauf, R.F. 1990. The Healing Forest: medicinal and toxic plants of the northwest Amazonia. Edited by: Theodore R. Dudley. Dioscorides Press. Portland, pp.484. Sielecki, A.R. , Fedorov, A. A., Boodhoo, A. , Andreeva, N.S. and James, N.G. 1990. Molecular and Crystal Structures of Monoclinic Porcine Pepsin Refined at 1.8 A Resolution. Journal of Molecular Biology 214: 143-170. Silva, D.H.C., Davino, S . C , de Moraes Barros, S . B. And Yoshida, M. 1999. Dihydrochalcones and Flavonolignans from Iryanthera lancifolia. Journal of Natural Products 62:1475-1478. i Silva, D .H .C , Pereira, F . C , Zanoni, M. V. B. and Yoshida, M. 2001. Lipophyllic antioxidants from Iryanthera juruensis fruits. Phytochemistry 57: 437-442. 152 Shahat , A . , C o s , P. , De Bruyne, T., Apers , S . , H a m m o u d a , F., Ismail, S . , A z z a m , S . C l a e y s , M. , Goovaer ts , E. Pieters, L , Vanden Berghe, D. A n d Vliet inck, A . J . 2002. Antiviral and Antioxidant Activity of F lavonoids and Proanthocyanid ins from Crataegus sinaica. Planta Medica 68: 534-538. Stermitz, F .R. , Lorenz , P. , Tawara , J . N . , Zenew icz , L.A. and Lewis , K. 2000. Synergy in a medic inal plant: Ant imicrobial act ion of berber ine potentiated by 5'-methoxyhydnocarp in, a multidrug pump inhibitor. Proceedings of the National Academy of Sciences of the United States of America 97: 1433-1437. Supuran , C.T . , S c o z z a f a v a , A . , C la re , B.W. 2002. Bacter ia l P ro tease Inhibitors. Medicinal Research Reviews 22: 329-372. Takech i , M. , Tanaka , Y . , Takehara , M. , Nonaka , G - l . A n d Nish ioka, I., 1985. Structure and Ant iherpet ic Activity A m o n g the Tann ins . Phytochemistry 24: 2245-2250. Vie i ra, P . C . , Y o s h i d a , M. , Gottl ieb, O .R . , Paul ino Fi lho, H. F., N a g e m , T . J . and Braz Filho, R. 1983. y Lactones from Iryanthera spec ies . Phytochemist ry 22: 711-713. 153 Xiong, Q.; Fan, W.; Tezuka, Y. ; Adnyana, I.; Stampoulis, P.; Hattori, M.; Namba, T.; Kadota, S. 2000. Hepatoprotective Effect of Apocynum venetum and its Active Constituents. Planta Medica 66: 127-133. Zhu, Phillipson, D., Greengrass, P.M., Bowery, N. and Cai , Y . 1997. Plant Polyphenols: Biologically ^Active Compounds or Non-Selective Binders to Protein? Phytochemistry 44: 441-447. 154 Chapter 6 Conclusions Obscurum per obscurius Ignotum per ignotius (The obscure through the more obscure, the unknown by the more unknown) Alchemica l max im 6.1. Search for Antibiotics in Medicinal Plants In the course of this project I have isolated, purified and structurally identified seven compounds from Piper lanceaefolium, which is traditionally used in the S ibundoy Va l ley (Colombia) for the treatment of a skin morbid condition caused by the "urine of the rainbow". T h e s e compounds are fairly inactive against micro-organisms such as yeast (C. albicans) and bacter ia (S. aureus). Their lack of potency thus prevents m e from consider ing them a s antibiotics. A role for these compounds in nature and their effect in human health has yet to be determined. In contrast, anti-viral and anti-bacterial activit ies were found for compounds isolated from Iryanthera megistophylla ( Ming et al., 2002). Probably the most important result from a pharmacological perspect ive is the very potent activity of iryantherin K against methicil l in-resistant Staphylococcus aureus. Iryantherin K and its es tereo isomer iryantherin L a lso presented moderate activity to C. albicans secre ted aspart ic proteases. Iryantherins s e e m to be a very interesting type of f lavol ignans whose mechan isms of act ion remain to be 155 elucidated but powerful antioxidant properties might be related to their antibiotic and enzymatic inhibition activities. Traditional use of /. megistophylla in the treatment of cutaneous leshmaniasis warrants further research to study the effect of these compounds in this parasitic disease. It is probably time to ask whether the different activities found were the consequence of our premises^ regarding the uses of traditional plants. It is also time to ask why, every time we inquire about antibiotic activities as I did, the result seems to be affirmative. In a large screening for antibiotic activities in medicinal plants used by British Columbian native peoples, McCutcheon and co-workers (1992) found that 69 plant extracts out of 73 presented antibiotic activity. Reports of this sort are common in the literature but often these studies are not followed by the corresponding phyto-chemical analyses, which makes it difficult to assess the potency of individual compounds compared to the whole plant extract activity. I believe that most of these "antibiotic" activities are detected regardless of their traditional use because of a methodological bias. Also I think that these putative "antibiotic" activities do not reflect a plant's ecological role nor do they explain traditional medicinal uses. From the methodological point of view, the disk diffusion assay used i throughout this study has a drawback particularly dealing with plant extracts. Large concentrations of plant extract are generally used, which are not realistic and certainly greater than the ones expected in a plant. It is also possible to 156 increase the number of apparently active extracts by selectively using particular bacteria or fungi that are very susceptible to chemical plant extracts. These two factors together may make the disk diffusion assay a unreliable methodology that affords many false positives and in consequence meaningless results. The fundamental question is then whether the likelihood of finding antibiotic compounds is increased by using ethno-botanical criteria instead of a random plant collection. In this regard it is interesting to note that plants collected within different medicinal categories (general tonic, non antibiotic medicine, related medicinal species and unspecified medicine) also presented a good portion of plants with antibiotic activity. Eighteen extracts out of a collection of twenty-three turned out to be active giving a success rate of 78% which seems to be a very high proportion considering that these plants are not traditionally used as "antibiotics". Furthermore, two out of four plants under the category non-medicinal, had antibiotic activity (McCutcheon et al, 1992). Interestingly the authors interpret this data as "indicative of the loss or distortion of knowledge concerning the specific uses of a given plant' (Mc Cutcheon et al, 1992). Rather than examining the biological meaning of the screening, the authors assumed a disruption in the traditional knowledge. This is not the first time that these types of concerns have been raised and alternative "experiments" have been proposed. For instance, it has been suggested to perform a "random" collection of plants and analyse their antibiotic activities and compare them with those traditionally used by a given community. 157 This proposition assumes that nature is a neutral field in the absence of human influence. It does not acknowledge the actual and/or past role of culture in shaping of the landscape. This culture/nature divide is the centre of great deal of academic activity and its implications are profound, but are well beyond the aim of this discussion. Suffice to say I do not hold any hope in finding such a thing as a random collection of plants, as Haraway (1992) pointed out "nature is a semiotic place". The representation of nature as the untouched wilderness is cherished by most Westerners and its political significance has been discussed elsewhere (Willems-Braun, 1997). However, whether this is testable or not I do not think is worth putting effort into finding a "random" collection of plants in the so-called "wilderness". Suffice to say that antibiotic screenings are not serving their purpose as "sieves" or "indicators" of extracts with antibiotic activity. Regardless of the potency of antibiotic activity found in each of these compounds, I believe that one should continuously bear in mind multiple effects of phenolic compounds in organisms. Closer attention must be paid then to the host-pathogen interaction during an infection event. For instance, quinine (present in Cinchona officinalis) and chloroquinine, exert their effects by inhibiting host - encoded functions (Ridley, 1997). This of course brings about the challenge of dealing with complex systems. The effect of a single compound or group of them at the cellular level in organisms (eukaryote or prokaryote) is complex and difficult to model. The concept of cause and effect is blurred in networks where feedback mechanisms 158 are the rule. Events occurring simultaneously are more likely to reflect reality better than cascade of events in a timely manner. In teasing apart these interactions, there is no obvious path to be followed and one should be able to overcome the horror that produces the mere idea of what complexity implies for our cause-effect mindset. In other words we need to develop intellectual tools that will allow us to look at the whole system with well-identified players. This implies the capacity to detect relevant cellular events without falling into simplified models that too often reflect our own limitations rather that a "real" phenomenon. There is a more sensitive issue that deals with the way we approach different cultures. This issue is particularly difficult to tackle for it implies an examination of the scientific community as a culture. This challenging undertaking will compel us to look differently at other's healing practices. 6.2. Understanding other's Medical Practices Very little attention has been paid (this thesis is an example) to the preparation and the cultural context in which a given plant is used as a therapeutic agent. We need to do a better job of studying traditional practices and in i understanding traditional therapeutical practices. We, must keep in mind for instance that "infection" is not a universally accepted category and recognise its cultural relativity as a western concept deeply rooted in the germ theory. It is intriguing to see how traditional knowledge is continuously praised as an 159 example of keen observation of nature and as a reflection of a profound knowledge of biochemical properties present in plant extracts without considering cultural contexts of illness. Many important biologically active compounds have been isolated from plants traditionally used in different parts of the world. However it is not uncommon to find bioactive compounds with activities that do not have evident correlation with their use in traditional societies. Lowering blood pressure effects of reserpine have not been studied in relation with Rauvolfia serpentina traditional use as a coadjuvant in reaching spiritual enlightenment. P. lanceaefolium is a case in point. It is not a minor task to correlate the chemical nature of these compounds with a cryptic traditional use associated with the moon cycle and the appearance of the rainbow. In this particular case one can observe how the moon cycle has traditionally been associated with crop rotation and in consequence with the whole subsistence in agricultural societies. It is likely these cycles are associated with insect pests that can eventually act as vectors of infectious diseases such as malaria or leishmaniasis. The genus Piper has been found to have important insecticidal chemical principles. The presence of taboganic acid and its action as an insect repellent might be an indication of possible role of these compounds in this regard. Although these hypotheses have not been tested yet I believe these alternatives are more likely than a putative antibiotic activity whose potency has been shown to be very low. Arguments over rationality and science endeavour 160 are expec ted . However these undertakings are at the very core of what the role of e thno-pharmacology should be as a discipl ine. If one des i res to understand the true use of medic inal plants by different cultures it is crucial to shift our mindset and be creat ive in generat ing hypotheses that take into account others' beliefs no matter how chal lenging the task might appear . \ A l s o , and probably more fundamental , is the need for the scientif ic community to real ise that medic ines (substances used in the treatment of s ickness) have culturally def ined meanings as wel l as b io-chemica l propert ies that endow them with speci f ic qualit ies and powers (Reyno lds and V a n D e r Gees t , 1988). The task of uncover ing the mean ing of med ic ines is not a trivial one. W e should be able to accompl ish this by looking in more detai l into the humoral theories of d i sease in traditional soc ie t ies a s wel l a s assimi lat ing the idea that therapy is about restoring a ba lance between contrast ing symbol ic qualit ies (Etkin, 1988), In this regard assess ing the eff icacy of natural remedies is highly problematic because of different understandings. For instance, Nor th-Amer ican indigenous people traditionally use plants known to be irritant and have vesicant use, which is not easi ly understood by westerners. However , the inducement of irritation, resulting in change of skin color and ves icat ion, is the physica l ev idence that d i sease entities are encounter ing the sur face to leave the body. Treatment and heal ing are then processual and only a series of ou tcomes will fulfill expectat ions (Etkin, 1988). 161 Thus efficacy as cultural construct is not necessarily consistent with biomedical criteria so when one cannot make sense of a plant use one should be wary in invoking the Doctrine of Signatures as an alternative exegesis. Concepts like "validation of traditional knowledge" illustrates western pretensions in accommodating indigenous knowledge only in western terms and advocating the right to dismiss other uses as irrational under the realm of superstition. Similarly we need to revisit the way we are drawing from indigenous knowledge and decide whether we wish to bridge cultures or we are just pragmatically interrogating traditional uses in order to increase efficiency in a long-term molecular farming project. At the beginning of the twenty-first century, research in medicinal plants used by traditional societies opens up a yet to be established path towards a better understanding of the role of herbalists (yerbatero) in a post-modern society. 162 6.3. References McCutcheon. A.R., Ellis, S .M. , Hancock, R.E.W. and Towers, G.H.N. 1992. Antibiotic screening of medicinal plants of the British Columbian native peoples. Journal of Ethnopharmacology 37: 213-223. Etkin, N.L. 1988. Cultural Constructions of Efficacy. In: The context of medicines in developing countries. Edited by: Reynolds, S. and Van der Geest, S. Kluwer Academic Publishers. Dordretcht. pp. 313. Haraway, D. 1992. The Promises of Monsters: A Regenerative Politics for Inappropriate/d Others. In: Cultural Studies. Edited by: L. Grossberg, C. Nelson and P. Treicher. New York: pp. 275-332. Ming, D.S., Lopez, A., Hillhouse, B.J. , French, C . J . , Hudson, J .B. and Towers, G.H.N. 2002. Bioactive Constituents from Iryanthera megistophylla. Journal of Natural Products 65: 1412-1416. Reynolds, S. and Van der Geest, S. 1988. Medicines in context. In: The context of medicines in developing countries. Kluwer Academic Publishers. Dordretcht. i pp.3-11. 163 Ridley, R. G. Dorn, A.g. , Vippagunta, S.R., Vennerstrom D. 1997. Haematin (haem) polymerization and its inhibition by quinoline antimalarials. Annals in Tropical Medicine and Parasitology 91: 559-566. Willems-Braun, B. 1997. Buried Epistemologies: The Politics of Nature in (Post)colonial British Columbia. Annals of the Association of American Geographers 87: 3-31. 164 165 166 0) E •o o (0 o o a> re a) o c re o o >» O >-CO o a E " X CO 5 Q 0 f Z o o Lu ^ 2 CL g S < v> *i a> co Q; a> o 0) c CO re re a> o 3 z X T -re c o '35 c E i5 6 IT) LU 4 -J Q. a. 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