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Antiviral activities of selected Chinese medicinal plants Yip, Lynn 1993

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ANTIVIRAL ACTIVITIES OF SELECTED CHINESE MEDICINAL PLANTSbyLYNN YIPB.Sc. Stanford University, 1978M.Sc. Stanford University, 1978A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESBiology ProgramWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAAugust, 1993© Lynn Yip, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of The University of British ColumbiaVancouver, CanadaDate ^t8 A1A-DE-6 (2/88)AbstractMedicinal plants in Yunnan Province of China were collected and screened forantiviral activity. Plants that were used to treat diseases that are now known to have viralcauses were selected through a systematic survey of information on traditional Chinesemedicine and the traditional medicines of ethnic minority groups in the region. Extracts from31 species in 22 plant families were assayed for inhibition of Sindbis and murinecytomegalovirus infections in mammalian cell cultures. Sixteen of the species showedantiviral activity. Elsholtzia ciliata (Thunb.) Hyland of the mint family (Lamiaceae) showedthe highest activity. It has more than one active component and one of them was purifiedusing bioactivity-guided phytochemical fractionation. The compound was identified as thepolycyclic aromatic hydrocarbon fluoranthene and its activity was enhanced with exposure tolong wavelength ultraviolet radiation. It has not been previously reported to have antiviralactivity.Investigations of the mechanism of action were carried out with the knownphotosensitive antiviral compound hypercin found in medicinal plants of the genusHypericum (Hypericaceae). Three hypericin derivatives and five related quinones weretested in structure-activity relationship studies. The new derivative 2,5,9,12-tetra-(carboxyethylthiomethyl) hypericin showed potent photosensitive virucidal activity againstmembrane-enveloped viruses. The photoaction was demonstrated to be of the singletoxygen type that could be reduced by the presence of a singlet oxygen scavenger. Incomparisons of mechanisms of action with that of hypericin in the presence and absence oflight, the two compounds showed similar potencies in light but hypericin was more potent inthe dark. Examination of the effect of these compounds on Sindbis virus structural proteinsshowed that treatment with the derivative in light caused an alteration of the capsid protein,an effect that was not shown in treatments with hypericin.Table of ContentsAbstract^  iiTable of Contents^  iiiList of Tables  viiList of Figures^  viiiAcknowledgements  ixGeneral Introduction^  1References^  6Chapter I.^Collection of Plants for Antiviral Screening UsingEthnopharm ac ologic al InformationIntroduction^  8Material and MethodsSelection of plants to screen for antiviral activity^ 13Plant collection^  16Crude extract preparation^  16Results^  17Discussion  27References^  31Chapter II.^Screening for Antiviral ActivityIntroduction^  33Material and MethodsCell culture^  37Cytotoxicity assays ^  38Assay viruses  38Antiviral screening assays^  38ivPlaque reduction assays^  40UVA irradiation  42Antibiotic assays^  42ResultsAntiviral activity screening^  42Activity of Elsholtzia ciliata and other Elsholtzia species ^ 44Antibiotic assays^ 46Discussion^  47References  52Chapter III. Purification and Identification of Active Component from Elsholtzia ciliataIntroduction^  56Material and MethodsAntiviral bioassays^  60Crude extract preparations  60Chemical separationsLiquid-liquid partition chromatography^ 61Flavonoid extraction^  61Column liquid chromatography andThin layer chromatography^  61Charcoal separation  62High performance liquid chromatography^ 62Structural analysesUltraviolet spectroscopy^  62Infrared spectroscopy  63Mass spectrscopy^  63High resolution mass spectroscopy^ 63Proton nuclear magnetic resonance spectroscopy^ 63ResultsChemical separation and purificationLiquid-liquid solvent partitioning^  64Flavonoid extraction and charcoal separation^ 64Fractionation procedures^  65Structural identificationUltraviolet spectroscopy^  70Infrared spectroscopy  72Mass spectroscopy^  73High resolution mass spectroscopy^ 73Proton nuclear magnetic resonance spectroscopy^ 74Antiviral activity of fluoranthene^  76Discussion^  77References  85Chapter IV. Structure-Activity Relationship Studies of the Antiviral Compound HypericinIntroduction^  88Photosensitizer Compounds^  89Hypericin^  92Material and MethodsChemicals^  96Antiviral assays  100ResultsScreeening of quinonoid compounds^  100Fagopyrum extract assays^  101Fungal compound assays  101Discussion^  104References  109vviChapter V.^Nature of Antiviral Action of the Hypericin Derivative EGK-149Introduction^  113Material and MethodsTime of treatment antiviral assays^  120Singlet oxygen mechanism assays  121Viral protein separation^  122ResultsTime of treatment activity assays^  122Singlet oxygen mechanism assays  125Effect of hypericin and EGK-149 on Sindbis virus proteins ^ 125Discussion^  129References  135Summary Discussion^  139AppendicesAppendix I. Medicinal plants assayed for antiviral activity and theirethnopharmacological indications for treatment^ 143Appendix II. Literature on the chemistry of Elsholtzia^ 145Appendix III. Infrared spectrum of purified fluoranthene^ 148Appendix IV. High resolution mass spectrum fragment analysisof purified fluoranthene^  149List of Tables1.1. Medicinal plants selected as candidates for antiviral screeningand their ethnopharmacological indications for treatment^ 181.2. Phylogenetic distribution of plants selected as candidatesfor antiviral screening.^ 262.1. Antiviral activity of crude extracts of Yunnan medicinal plants againstmurine cytomegalovirus (MCMV) and Sindbis virus (SINV)^ 432.2. Minimum active concentrations of methanolic fractions againstmurine cytomegalovirus (MCMV) and Sindbis virus (SINV)^ 452.3. Antibiotic activity of the crude extract of Elsholtzia ciliata 463.1. Species of Elsholtzia used in Chinese medicine.^ 583.2. Concentrations of solvent partition fractions active againstSindbis virus with UVA light^ 643.3. Extinction coefficients from the ultraviolet spectra of fluoranthene.^ 713.4. 1 H-NMR spectral assignments of fluoranthene^ 753.5. Minimum active antiviral concentrations of fluoranthene^ 764.1. Minimum active antiviral concentration of hypericin compoundsagainst Sindbis virus.^ 1024.2. Minimum acitive antiviral concentrations of Hypericumand Fagopyrum crude extracts.^ 1024.3. Minimum active antiviral concentrations of cercosporin^ 1034.4. Minimum active antiviral concentrations of Hypocrella compounds^ 1035.1 Percentage of Sindbis virus plaque reduction from treatmentsat different stages of viral infection cycle^ 1245.2 Percentage of murine cytomegalovirus plaque reduction fromtreatment with compound EGK-149 at different stages of viral infection cycle ^ 125vi iList of Figures1.1. Map of Yunnan Province, China  102.1. Plaque reduction assay of antiviral activity.^ 413.1. Illustration of Elsholtzia from a Chinese herbal. 563.2. Normal phase HPLC chromatograph of Elsholtzia fraction.^ 673.3. Separation of active compound by reverse phase HPLC. 693.4. Chemical structure of fluoranthene^ 703.5. UV spectrum of purified fluoranthene. 713.6. EI mass spectrum of purified fluoranthene^ 733.7. 1 H-NMR spectrum of purified (A) and authentic (B) samplesof fluoranthene.^ 744.1. Hypericin skeleton with carbon positions^ 934.2. Chemical structures of hypericin and derivatives 974.3. Chemical structures of bianthrones^ 974.4. Chemical structure of fagopyrin. 984.5. Chemical structure of duclauxin.^ 984.6. Chemical structure of cercosporin. 994.7. Chemical structures of hypocrellin compounds^ 995.1 Sindbis virus plaque reduction assay platesfrom treatments with compounds at different stages of viral infection cycle.... 1235.2. Percentage of Sindbis virus plaque reduction by compound EGK-149in the presence of cholesterol^ 1265.3 Percentage of Sindbis virus plaque reduction by hypericinin the presence of cholesterol 1275.4 Electrophoretic gel (SDS-Page ) of Sindbis virus proteinstreated with compound EGK-149 and hypericin.^ 128vi i iAcknowledgementsMy gratitudes go first to my supervisor Dr. G.H.N. Towers who gave me the auspicesto conduct this research. His enthusiasm and hospitality have also been very muchappreciated. I thank Dr. J.B. Hudson for his guidance with a significant part of my workwhich was done in his laboratory, and the other members of my supervisory committee: Dr.F.S. Abbott, Dr. B.A. Bohm, Dr. G.C. Hughes and Dr. N.J. Turner for their valuable inputs.I am grateful to Professors Zhou Jun and Pei Shengji of the Kunming Institute ofBotany for sponsoring my research in China and for arranging the succeeding acquisition ofplant materials. I also thank the staff of the Ethnobotany Laboratory, as well as ProfessorsYang Zongren, Chen Siying and Zang Mu for their assistance. I am honored by the sharingof their knowledge from Drs. Fan Bingjun, He Zhegao and He shixiu. For help with plantcollecting I thank Wang Zongyu, Xu Hua, Shi Zhaolong and Cui. Jingyun. I thank Lu Lipingand Zhang Yen for accompanying me on collection trips and appreciate their friendships.For providing chemical compounds that were tested as part of my studies, I givethanks to Dr. E. Gruszeka-Kowalik and Dr. D. Zembower from the laboratory of Dr. L.Zalkow; Dr. M. Daub, Dr. J. Jacyno, Prof. J. Zhou. and Dr. J. Kagan. I also thank the officersof the Program for Collaborative Research in the Pharmaceutical Sciences at the Univeristyof Chicago for providing the NAPRALERT data search. For technical assistance, I thankElizabeth Graham for her help with antiviral assays; Chantal De Soucy-Brau, Dr. Terry Jarvisand Marietta Austria for the operation of IR, EI/MS and NMR instruments; and AllisonMcCutcheon and Shona Ellis for the use of their antibiotic assay system. All members of theBotany Department office staff are appreciated for their helpfulness.Members of Dr. Towers' laboratory have provided invaluable support. I would like toespecially thank Felipe Balza for his expertise in chemistry, and Zyta Abramowski for alwaysbeing so helpful with her knowledge and experience. Fellow graduate students and researchfellows have helped me with good discussions and comraderie. I would like to thank inparticular Shona Ellis, Dr. Peter Constabel, Dr. Paul Spencer, Dr. Hector Barrios-Lopez, Dr.Fumito Ichibashi and Dr. John McCallum.Finally, I am most grateful to my parents for having always encouraged and supportedme in every way in my pursuit of knowledge.ixGeneral IntroductionThe goal of this project was to conduct a comprehensive study of biologically activecompounds from selected plant species, encompassing aspects of ethnophannacology, botany,microbiology, chemistry and pharmacology. The study included: a) the screening of selectedmedicinal plants for bioactivity, b) the chemical purification of active compounds, and c) thedetermination of the nature or mechanism of the activities. The introduction is a perspective onthe uses and usefulness of ethnopharmacological information, from traditional medicine, in ascreening program for bioactive compounds. The general term 'pharmacognosy' describes thesearch for bioactive compounds when the context of the research has the potential fordeveloping new therapeutic chemicals. This phase of the study can also be considered as partof ethnobotany since it is a screening of plants used by indigenous peoples. Ethnobotany hasbeen defined by Ford (1978) as "the study of direct interrelations between humans and plants".Ethnopharmacology is more specifically defined by Bruhn and Holmstedt (1981) as "theinterdisciplinary scientific exploration of biologically active agents employed or observed byman".The study of biologically active compounds is a basic part of pharmacology. Thepharmacology of traditional medicines is based on the uses of natural products used by culturalgroups in the treatment of disease and this body of knowledge can be viewed as the historicalresult of empirical testing by practitioners of folk medicine. It is my view that this research canprovide guides in the search for bioactive natural compounds. The significance ofethnopharmacological research in discovering new therapeutic activities of natural products hasbeen extensively discussed (Schultes and Swain, 1976; Bruhn and Holmstedt, 1981; Delaveau,11981; Malone, 1983; Kyerematen and Ogunlana, 1987; Cox, 1990; Farnsworth, 1990).According to Farnsworth et al. (1985) 74% of the 119 bioactive plant-derived compoundscurrently in world-wide use were identified via research based on leads from folk or ethno-medicine. Although historical evidence indicates that traditional medicine has provided goodleads to therapeutic chemicals, the question still arises as to whether an ethnopharmacologicallyguided screening is more effective than random sampling in yielding active compounds.Several comparative studies (Spjut and Perdue, 1976; Verpoorte, 1989; Balick, 1990) supportthe validity of the approach of using ethnopharmacological information as opposed to randomscreening.The information traditional medicine can provide depends on the objective of thescreening program and the treatment of the original data. It is important at this point toconsider what the ethnopharmacologist should be aware of in the process of eliciting guides tobioactivity screening from traditional medicine information. I use the term 'traditionalmedicine' to mean a body of historically and culturally transmitted knowledge of medicinebased on theories of health and disease. Traditional medicines have culturally based views andprinciples regarding diagnosis and cure of diseases that may not be directly matched withwestern scientific research concepts (Elisabetsky, 1986). The uses of plants for healing canoften be based on perspectives very different from what the screening program is based on.Therefore, there is an important element of interpretation involved in doing ethno-pharmacological research. First of all, the information has frequently to be translated fromanother language. The interpretation of the meaning of terminology may require anunderstanding of the framework that they come from, and the treatment of information couldcall for some careful bridging and correlating of different perspectives.In studying the activity of chemicals from traditional medicinal plants, there aredifferent extents of correspondence between the effects found or assayed for, and what the2plant was used for. It is a commonly encountered thought that this type of study is testing theefficacy of traditional medicines. There are a number of cases where this is done to somedegree because the activity of the isolated plant compound shows a direct reflection of how theplant was used. Morphine, an alkaloid from the opium poppy plant (Papaver somniferum L.)that was used to induce euphoria, has been found to be an analgesic that exerts its effect bybinding to specific neural receptors (Evans et al. 1988). Similarly, tubocurarine, which wasderived from plants of the Loganiaceae used as arrow poisons, has been shown to be effectiveas such by being a potent muscle relaxant (Bryn Thomas, 1963). Areas where the treatmentspracticed by both modern and traditional methods are more likely to be aimed at the sametargets would probably show higher correlations between ethnopharmacological indicationsand activity screening results. For example, whether plants used to treat topical infections haveantibiotic activity can be ascertained by in vitro antibacterial bioassays. Other important drugsfrom plants, however, have activities that are not directly indicated by their usage in traditionalmedicine.The anticancer agents taxol from the Pacific yew tree (Taxus brevifolia Nutt.), andvinblastine and vincristin from the Madagascar periwinkle Catharanthus roseus (L.) G. Don.,were all discovered in laboratory screening programs (Farnsworth, 1984). Even though theseplants have been used in traditional medicines, they were not used to treat cancers. There maynot be many direct ethnopharmacological indications for cancer treatment due to the complexityand variety of cancers which are being elucidated by western medicine. The number of distinctcancers is estimated at over 100 and up to 300, with no apparent common denominator to thedifferent types (Suffness and Pezzuto, 1991). What can now be diagnosed as cancer may havebeen more likely diagnosed in folk medicine as an ailment in a particular body organ rather thanspecifically as a cancer. The discoveries of these drugs demonstrate that medicinal plantspresent good sources for systematic western scientific screening, even though the target of thescreening may not be well indicated due to conceptual differences between the traditional3diagnosis of illnesses and the aims of modern screenings. The chances of finding bioactivecompounds are probably higher when there is good correlation between the screening targetand traditional usage. However, chances of finding bioactive compounds from a generalscreening of plants that have histories of being used medicinally are also probably higher than arandom screening of plants per se.Western medical research has also elucidated the microbial agents responsible for manydiseases. The importance or relevance of finding new antiviral compounds comes from the factthat although over 400 viruses have been identified in the pathogenesis of a wide range ofhuman diseases (Choppin, 1986), very few antiviral compounds have made their way toclinical use (Prusoff, 1988). The factors that contribute to the slowness in development ofantiviral drugs were discussed by Galasso (1988). One of the major challenges in findingeffective antiviral compounds is due to the intimate biochemical association between the virusand its host cell, such that it is difficult to damage the virus without damaging its host (Walker,1988). This close relationship between the virus and cell, however, also makes antiviralcompounds potentially useful tools in research on the nature of cells and viruses. Manyantiviral compounds have been isolated from medicinal plants (Vanden Berghe et al. 1978;Hudson, 1990), but the applicability of some of them as potential drugs is still at preliminarystages of development and the phytochemical search for antiviral compounds will continue tobe a growing area of research (Vlietinck and Vanden Berghe, 1991).I have presented here the reasoning and considerations for using anethnopharmacological approach to search for antiviral compounds in medicinal plants. Themethodology of research on medicinal plants is usually divided into a series of stages involvingthe synthesis of work from the disciplines of pharmacognosy, botany, chemistry andpharmacology (Cave, 1986). It was the goal of this research to conduct a project thatencompasses such a synthesis by incorporating the following stages:41. Systematic search of traditional medicine information toselect plants for antiviral screening.2. Collection and taxonomic identification of plants.3. Bioassay screening for antiviral activity.4. Purification of active component(s).5. Investigation of nature of activity.Researchers of bioactive plant compounds have frequently expressed the need for goodintegration between the different disciplines involved, since aspects of the work are often doneby different specialists. The best way to understand the interfaces between the differentdisciplines necessary for strong integrated research is to do a comprehensive investigation thatincludes all the aspects. It is hoped that good understanding of the research involved inexploring the bioactive natural compounds will contribute to the beneficial management of arich resource. This is particularly pertinent in view of the fact that we are faced with the loss ofboth biotic and cultural resources in an era of tremendous social and environmental change.5ReferencesBalick, M.J. (1990) Ethnobotany and the identification of therapeutic agents from the rainforest. In: Bioactive Compounds from Plants. Wiley, Chichester (Ciba Found. Symp.154), pp. 22-39.Bruhn, J.G. and Holmstedt, B. (1981) Ethnopharmacology: Objectives, principles andperspectives. In: J.L. Beal and E. Reinhard (eds.), Natural Products as MedicinalAgents. Hippokrates Verlag, Stuttgart, pp. 405-430.Bryn Thomas, K. (1963) Curare, Its History and Usage. J.P. Lippincott, Philadelphia,144 pp.Cave, A. (1986) Methodology of research on medicinal plants. In: D. Barton and W.D.(eds.) Advances in Medicinal Phytochemistry. John Libby, London, pp. 47-57.Choppin, P.C. (1986) Basic virology. In: In: H. Rothschild and J.C. Cohen (eds.) Virology inMedicine. Oxford University Press, New York, pp. 3-45.Cox, P.A. (1990) Ethnopharmacology and the search for new drugs. In: BioactiveCompounds from Plants. Wiley, Chichester (Ciba Found. Symp. 154), pp. 40-55.Delaveau, P. (1981) Evaluation of traditional pharmacopoeias. In: J.L. Beal and E. Reinhard(eds.). Natural products as medicinal agents. Hippocrates Verlag, Stuttgart, pp. 395-404.Elisabetsky, E. (1986) New directions in ethnopharmacology. J. Ethnobiol. 6, 121-128.Evans, C.J., Hammond, D.L., and Frederickson, R.C.A. (1988) The opiate peptides. In:G.W. Pasternak (ed.) The Opiate Receptors. Humana Press, Clifton, pp. 23-71.Farnsworth, N.R. (1984) Medicinal plants and drug development. In: P. Krogsgaard, S.B.Christensen and H. Koford (eds.) Natural Products and Drug Development. (AlfredBenzon Symp. 20), Murksgaard, Copenhagen, pp. 17-28.Farnsworth, N.R. (1990) Ethnopharmacology and drug development. In: BioactiveCompounds from Plants. Wiley, Chichester (Ciba Found. Symp. 154), pp 2-21.Farnsworth, N.R., Akerele, 0., Bingel, A.S., Soejarto, D.D. and Guo, Z. (1985) Medicinalplants in therapy, Bull. of the World Health Organization 63 (6), 965-981.Ford, R.I. (1976) Ethnobotany: historical diversity and synthesis. In: R.I. Ford (ed.) Thenature and status of etnobotany. University of Michigan, Ann Arbor, pp. 33-49.Galasso, G.J. (1988) Promises to keep: clinical use of antiviral drugs. In: E.De Clercq (ed.)Clinical Use of Antiviral Drugs. Martinus Nijhoff, Boston, pp. 387-403.Hudson, J.B. (1990) Antiviral Compounds from Plants. CRC Press, Boca Raton, 200 pp.Kyerematen, G. A. and Ogunlana, E.O. (1987) An integrated approach to the pharmacologicalevaluation of traditional materia medica. J. Ethnopharm. 20, 191-207.6Malone, M. (1983) The pharmacological evaluation of natural products - general and specificapproaches to screening ethnopharmaceuticals. J. Ethnopharm. 8, 127-148.Prusoff, W.H. (1988) Idoxuridine, or how it all began. In: E.De Clercq (ed.) Clinical Use ofAntiviral Drugs. Martinus Nijhoff, Boston, pp.15-23.Schultes, R.E. and Swain, T. (1976) The plant kingdom: a virgin field for new biodynamicconstituents. In: N.J. Fina (ed.) The Recent Chemistry of Natural Products, IncludingTobacco. (Proceedings of the Second Philip Morris Science Symposium), New York,pp. 133-171.Spjut, R.W. and Perdue, R.E. Jr. (1976) Plant folklore: a tool for producing sources ofantitumor activity? Cancer Treatm. Rep. 60, 979-982.Suffness, M. and Pezzuto J.M. (1991) Assays related to cancer drug discovery. In: BioactiveCompounds from Plants. Wiley, Chichester (Ciba Found. Symp. 154), pp. 71-134.Vanden Berghe, D.A., Ieven, M., Mertens, F. and Vlietinck, A.J. (1978) Screening of higherplants for biological activites: II. Antiviral activity. Lloydia 41, 463-471.Verpoorte, R. (1989) Some phytochemical aspects of medicinal plant reserach.J. Ethnopharm. 25, 45-59.Vlietinck, A.J. and Vanden Berghe, D.A. (1991) Can ethnopharmacology contribute to thedevelopment of antiviral drugs? J. Ethnopharm. 32, 141-153.Walker, R.T. (1988) Antiviral chemotherapy: an introduction and reasons for the slowprogress, particularly towards rational design. In: E. De Clercq and R.T. Walker(eds.) Antiviral Drug Development: A Multidisciplinary Approach. NATO ASI SeriesA, Vol. 143, Plenum Press, New York, pp. 1-10.7Chapter I.Collection of Plants for Antiviral ScreeningUsing Ethnopharmacological InformationIntroductionMedicinal plants in Yunnan Province, People's Republic of China, were selected forantiviral screening using information from traditional medicines. China has one of the oldestcontinuous and documented systems of traditional medicine. In folklore, the practice of usingherbs in healing is attributed to the efforts of the semi-mythological emperor Shen Nongapproximately five thousand years ago. There is a written herbal called the Shen Nong BenCao Jing that is dated to about 200 A.D. (Huang, L.,1984). The most comprehensive historiccompendium of the Chinese medicinal herbs was compiled by Li Shi-zhen in the Ming dynastyin 1578 A.D.. This materia medica, the Ben Cao Gang Mu contained 1,892 entries (Li,1979). An appendix was made later by Zhao Xue-min with 716 more items. A computerdatabase at the Chinese University of Hong Kong lists 4,941 species of higher plants from thetraditional Chinese pharmacopoeia (Duke and Ayensu, 1985). The recorded use of these plantspresents a large source of organized ethnopharmacological information. In addition, there areover 20 ethnic minority groups in Yunnan. These minority groups include ones like the Daiand the Karen that are of Southeast Asian affinity, and ones with Tibetan links. These groupshave established practices of traditional medicine and many of them have historic documents intheir own languages. The Dai have their own Institute of Traditional Medicine in Jinghong,where the medicine is practiced as well as researched. Near the town of Dali, there is an annualgathering where people of many different groups travel from long distances to trade, and8medicinal materials are a significant commodity (Towers, personal communication).Ethnobotanical information from the minority groups is in the process of being recorded and itis also translated to Chinese (Yunnan Institute of Medicine Inspection, 1983). The medicinalplants used by these people adds to the pool of candidates for screening. Six hundred andseven plant species are listed in the Chinese Medicinal Plants of Yunnan (Kunming ReserveUnit Health Division, 1970) and its sequel volume (Kunming Institute of Botany, 1978).Yunnan Province in the southwest of China (lat. 21'10-29'00 N, long. 97'30-106'10E), is an area known for its botanical diversity (Pei, 1988). The diversity is probably a resultof the variety of geo-climatic conditions found in the province. A map showing the location ofthe province is provided in Figure 1.1. The southern-most region is tropical and includes theXishuangbanna Prefecture which is reputed to have many unusual and endemic organismsbecause of the geography of being surrounded by a ring of mountains. Zou (1988) describedthe composition and features of the Xishuangbanna flora. Over 3800 species of higher plantshave been identified in Xishuangbanna, and this number was estimated to represent one sixthof the Chinese flora (Yunnan Institute of Tropical Botany, 1984). The western part of theprovince has increasingly temperate to alpine conditions due to both gradual and dramatic risesin elevation (Zhang, 1983). There is a plantation where medicinal plants are cultivated in theCang mountains outside Dali. The diverse flora of this region has also drawn attention from anumber of botanical expeditions (Lancaster, 1981; Grey-Wilson, 1988). The rich resources inboth plants and ethnobotanical knowledge made Yunnan an ideal area for conducting ascreening program.910Figure 1.1: Map of Yunnan ProvinceThe literature of the herbal pharmacopoeia is usually organized by plant names. Thereare also collections of herbal mixture recipes for the treatment of particular symptoms andillnesses. The Chinese names of the herbs are common names. For this survey, only theliterature which includes the corresponding Latin scientific names was used. It wasnonetheless necessary to be aware that it is common for a Chinese name to have been applied tomore than one species. In many cases, these are very similar species of the same genus thatcould likely be disputable taxonomic entities. Distinct species or even species in differentgenera are sometimes given the same name. In some instances because they are found indifferent regions, they are distinguished with prefixes denoting geographic origins. In manycases the common base name parallels a generic name, and further differences are againdenoted by descriptive prefixes. In the uses of the plants medicinally, sometimes only the basename is specified because the different species or varieties are considered to be of the samenature and therefore similarly applicable. It must be remembered that the traditional practice ofherbal medicine in China was not developed using the Linnaean system of taxonomicclassification. Yet, I find that there is quite an impressive correlation between the traditionalChinese and Linnaean classifications. Credits are due to both the acute observations that formthe basis of traditional knowledge, and the work that has gone into correlating that knowledgewith the scientific classification system.The documentation of a Chinese medicinal plant usually includes the followinginformation: 1) name and other common names,2) morphological characteristics,3) habitat,4) part used and collection procedure,5) nature of medicine,6) uses in treatment,7) dosage, methods of preparation and mixture recipes.1 1The uses in treatment often encompass quite a large array of different symptoms andsicknesses. As an example, the pan-tropical weed Bidens pilosa L. (Asteraceae) is cited foruse in the treatment of : cold, prevention of influenza, bites by poisonous snakes and insects,hepatitis, bladder infection, intestinalitis, dysentery and rheumatoid arthritis in Yunnan ChineseMedicinal Plants (Yunnan Reserve Health Unit, 1970). In the view of traditional Chinesemedicine, this apparently diverse range of maladies has certain common characteristics that areaffected by the nature of the medicinal herb. The interpretation of what these maladies have incommon is based on a theoretical framework of health. The components of the body,medicines and illnesses are all viewed from the perspective of a balance of yin and yangnatures. The yin natures are passive, cold and wet and the yang natures are active, hot anddry. The yang nature is associated with the skin, bones and musculature, and the yin naturewith the internal organs. The two natures are always coexistent in varying proportions. Theheart, for example, is considered yang dominant but has the yin component of an internalorgan. Medicines are used to restore the balance of natures disrupted by disease. A simpleexample of working with the principle of natures is when a medicine of a cold nature is used tocounter the hot nature of a fever, or an internal inflammation. Medicines are further designatedwith specific pharmacological properties (Huang, S., 1984). Many of these properties can bemore directly correlated with western pharmacological concepts. Some of these are: the abilityto increase circulation (stimulatory), to induce diuretic effects, to cure infections (antibiotic,anti-inflammatory), to calm (sedative), and to dispel parasitic worms (anthelmintic). Otherapplications are based on more holistic concepts such as detoxification, tonics and generalinvigoration.The traditional practice uses a combination of herbs to heal the affected organs as wellas achieve an amelioration of the balance of natures. The mixture of plants in a medicinalrecipe thus consists of principal acting and supplementary acting ingredients. This leads to thesituation that in the compilation of information on its usage, a plant often shows a number of12different applications because it may have been used as the primary ingredient or forsupplementary effects in different prescriptions. When addressing this information for leads tobioactivity, what can be used as an indication of what is likely the principal target of a plant'saction is to look for citations by more than one source for a particular indication. A plant that iscited for a more specific versus a wide range of uses, is also more likely to be targeted morespecifically in its application.To conduct an antiviral screening of the medicinal plants, I had to establish criteria forthe selection of plants for testing from this information base. The interpretation of theinformation can be complex because it is derived from an elaborate conceptual system of healthand medicine. For the purposes of a general search for antiviral activity, the informationregarded can nonetheless be obtained with a simpler approach. Some of the diseases that arenow linked to viral causes are frequently cited as being treated with medicinal herbs. Thesediseases are: the cold (rhinovirus), influenza, the flu (influenza virus), and hepatitis orsymptoms of liver disorder (hepatitis viruses). The causes of warts are also viral. Theapproach was thus to systematically survey the literature on the Yunnan pharmacopoiea forplants that have been used in the treatment of these diseases, and to collect the plants for ageneral screening of antiviral activity.Materials and MethodsSelection of plants to screen for antiviral activityAn initial list was compiled of Yunnan plants that have been used for the treatments ofviral diseases from the two volumes of the Chinese Medicinal Plants of Yunnan. The plants13on this list were surveyed for citation in other literature on the traditional Chinesepharmacopoeia. The literature sources surveyed were:A) in Chinese:1. Chinese Medicinal Plants of Yunnan. (1970)Kunming Reserve Unit Health Division, 777 pp..Chinese Medicinal Plants of Yunnan, Sequel. (1978)Kunming Institute of Botany, 535 pp..Tiangjing People's Publishing, Tiangjing.2. Yunnan Chinese Medicine Herbs. (1971)Yunnan Health Bureau Revolutionary Committee, 213 pp..Yunnan People's Publications, Kunming.3. Pharmacology of Chinese Medicinal Herbs. Vol. 2 (1980)Nanjing Institute of Traditional Medicine, 742 pp..Jiangsu People's Publications, Nanjing.4. Dictionary of Chinese Medicine. Vol. 1 and 2 (1977)New Jiangsu Hospital, 2754 pp. + 764 pp. appendix.Shanghai Science and Technology Publications, Shanghai.(the dictionary information is compiled from other sourcesand includes information from minority tribes)B) in English:5^Medicinal Plants of China. Vol. 1 and 2, (1985)Duke, J.A. and Ayensu, E.S., 705 pp.,Reference Publications, Algonac6. Herbal Pharmacology in the People's Republic of China. (1975)A trip report of the American Herbal Pharmacology Delegation, 269 pp.National Academy of Sciences, Washington, D.C..7. Chinese Medicinal Herbs. (1973)compiled by Li Shi-Chen, 482 pp.,translated from Chinese by F. Porter-Smith and G.A. Strand.Georgetown Press, San Francisco.8. Oriental Material Medica. (1986)Hsu, H.Y., Chen, Y.P., Shen, S.J., Hsu, C.S., Chen, C.C.,and Chang, H.C., 932 pp.,Oriental Healing Arts Institute, Long Beach.9. Pharmacology and Applications of Chinese Material Medica. (1986)Chang, H.M. and But, P.H. (eds.), Vol. 1 and 2,CMMRC, Chinese University of Hong Kong,World Scientific Publishing, Singapore.14The list of potential plants for testing was appended with the survey of a number ofpublications on the traditional medicine of Yunnan ethnic minority tribes, in Yunnan. Theseare usually small booklets, written in Chinese but some have notations in the minority tribelanguage. There are no specific authors of these booklets.a. Common Medicines of the Lagou Tribe (1987)Simao District Traditional Medicine Research Station.b. Dai Traditional Medicine Recipes (1985)Xishuangbanna Traditional Medicine Research Office.c. Ethnic Medicine of De Hong (1983)De Hong Prefecture Health Unit Drug Inspection Station.d. Ethnic Medicines of the Hani Tribe of Yuenjiang. (1978)Yuxi District Drug Inspection Station.e. Herbal Medicines of Lijiang. (1971)Lijiang District Revolutionary Committee Production Division Health Unit.f. List of Yunnan Minority Tribe Medicines. (1983)Yunnan Institute of Medicine Inspection.g•^Medicines of the Dai of Xishuangbanna. (1987)Vol. 1, 2, 3 and appendix to medicinal recipes,Xishuangbanna Prefecture Science and Technical Committee andXishuangbanna Prefecture Health Unit.h. Medicines of the Diqing Tibetans .(1987)Yunnan Minority Publications.i. Naxi Tribe Medicines (1979)Lijiang District Drug Inspection Station.j.^Yi Tribe Medicines (1979)Lijiang District Drug Inspection Station.Plants were prioritized in qualification for screening on the basis of specificity ofapplication and frequency of citation for similar uses in different sources of information. Aplant was considered to be a good candidate for collection if it did not have a large range ofother uses besides those previously mentioned. A plant was also of more interest if it was citedby more than one source for uses in the screening criteria. During the process of plant15collection, this prioritization was used as a guideline as to which plants to look for. Whichplants were actually collected was also largely determined by availability.Plant collectionPlant collection was made in Yunnan Province with the collaboration of the KunmingInstitute of Botany (MB) of the Chinese Academy of Sciences. Plants were collected fromthree regions of Yunnan during the months of July and August, 1988: Kunming area in centralYunnan; Dali-Lijiang area in northwestern Yunnan; and Xishuangbanna in the south of theprovince. In each region, local botanists assisted with the location and identification of plants(Wang Zongyu of the MB in Kunming, Xu Hua and Shi Zhaolong of the Dali Drug InspectionStation, Cui Jingyun of the Yunnan Institute of Tropical Botany in Xishuangbanna). Someplants were collected with the help of herbal doctors that currently use them (Dr. Fan Bingjunin Dali, Drs. He Zhegao and He Shixiu of the Naxi minority in Lijiang). Five samples werefrom plants already collected in preparation for use by Dr. He Shixiu. Two samples werepurchased from the pharmacy of the Dai Traditional Medicine Institiute in Jinghong,Xishuangbanna.Voucher herbarium specimens of the plant species collected have been deposited at theHerbarium of the University of British Columbia (UBC) in Vancouver. Identifications of thevoucher specimens were verified by Li Yenhui at the Ethnobotany Laboratory of the KunmingInstitute of Botany in Kunming, Yunnan.Crude extract preparationExtracts were made in Kunming from air-dried samples of the whole plant or, in a fewcases, from the portion specified as being used medicinally. Ten-gram samples of plant16material were percolated exhaustively in 90% ethanol and dried under reduced pressure atbelow 40° C. The extracts were dried to residues for ease of transportation back to UBC inVancouver for testing. Official permission was obtained for the export of these samples fromthe People's Republic of China.ResultsThe list of potential plants for antiviral screening is shown as Table 1.1. The viralillnesses for which the plants are used are also listed. The sources for the treatment citationsare shown by numbers and letters referring to the publications listed in the previous section.The publications on medicines of minority peoples are designated with letters and on traditionalChinese medicine designated with numbers. In some cases, the pharmacological indicationscited for a plant apply to two closely related species and they are listed together. There were 82species selected, from 69 genera and 43 plant families.The phylogenetic distribution of the plant families according to order and class isshown in Table 1.2.17Table 1.1Medicinal Plants Selected as Candidates for Antiviral Screeningand Their Ethnopharmacological Indications for TreatmentPlant Family^Species^ Part Used #Apiaceae^Centella asiatica (L.) Urban^**^PL(Umbelliferae)cold 1 , infectious hepatitis 1 , jaundice 4,hepatitis f, (5).Apocynaceae^Plumeria rubra var. acutifolia^**^RT(Poir.) Ball.hepatitis a , b , g, (4,f,5-herpes).Araceae^Alocasia macrorrhiza (L.) Schott^ RTcold 1 .Araliaceae^Livistona chinensis R. BR.^*^LF(Palmae)hepatitis b, (4).Araliaceae^Hedera nepalensis K. Koch^ PLhepatitis 1 .Asclepiadaceae^Dregea sinensis Hemsl.^*^PLhepatitis a.Asteraceae^Bidens pilosa L.^ **^PL(Compositae)cold 3, f, prevent flu 1, 4, 8 , jaundice 1 , (5).Blumea balsamifera (L.) DC.^ PLcold 1 , flu 1 , (4).Dichrocephala benthamii C.B. Clarke^* *^PLor D. chrysanthemifolia (B1.) DC.hepatitis 1, 2, f, cold 1, 4, (i).used by Dr. Fan Bingjun for cold1819Plant Family^Species^ Part Used #Asteraceae^Laggera pterodonta (DC.) Benth.^ PLCold 1, 3, 4 , prevent flu 1, (5). +used by Dr. Fan Bingjun for coldSpilanthes paniculata Wall.^ PLcold 1, 4,Berberidaceae^Mahonia nepalensis DC.^**^BRhepatitis b.used in 4 of 7 mixtures for hepatitis treatment.(purchased from the Dai Traditional MedicineInstitute at Jinghong, Xishuangbanna)Brassicaceae^Rorippa montana (Wall.) Small^ PL(Cruciferae)flu 1, 4, jaundice hepatitis 1 , warts 1 .Caesalpiniaceae^Cassia tora L.^ SDhepatitis 4, b, supports liver 6, 8 , (5,7).Combretaceae^Quisqualis indica L.^ **^PL,FRhepatitis b, (4,6,7,9)Convolvulaceae^Dichondra repens Forst.^**^PLflu f, jaundice hepatitis 1 , jaundice 4, 8 ,liver diseases 9 , (5).Cornaceae^Dendrobenthamii capitata (Wall.) Hutch.^LF,FL,FRhepatitis 1, 4 .Helwingia himalaica HK. f. et Thomas^LF,FLflu 1 , (4).Cycadaceae^Cycas siamensis Miq.^**^LFhepatitis A 1 , jaundice hepatitis 1 .Dillenaceae^Dillenia indica L.^ BKhepatitis b.20Plant Family^Species^ Part Used #Dipsacaceae^Dipsacus asper Wall.^ RTsupports liver 3, 4, 8, g, (1).Ebenaceae^Diospyros kaki L.f.^ **^BKjaundice hepatitis 8, f, (1,4,7).(purchased at the Dai Traditional MedicineInstitute at Jinghong, Xishuangbanna)Euphorbiaceae Breynia rostata Merr.acute hepatitis 1 , hepatitis 1 , cold 1 .PLEuphorbia prolifera Ehrenb. ex. Boiss.warts 1, f.** PLEuphorbia spp. were frequently cited forthe treatment of warts 1, 4, f.Homonia riparia Lour.infectious and chronic hepatitis 1, 4 .^+* PLRicinus communis L.jaundice hepatitis f, (1,4,7,8).** LF,RTFabaceae Desmodium triquetrum (L.) DC. * PL(Leguminosae)cold 4, 8, b, (2,5).Kummerowia striata (Thunb.) Schindl.cold 1 , infectious hepatitis 1,4 .used by Dr. Fan Bingjun for hepatitis** PLGentianaceae Halenia elliptica D. Donhepatitis 1, 2, f, h, (4).** PLSwertia punicea Hemsl.or S. yunnanensis Burkill** PL(common name = hepatitis herb)infectious and jaundice hepatitis 1,2 ,hepatitis 4, acute hepatitis 9 .^+21Plant Family^Species^ Part Used #Hypericaceae^Hypericum japonicum Thunb.^ PLinfectious and chronic hepatitis 1 ,hepatitis 3, 4, 6, 1, (a).Hypericum patulum Thunb.^ PLinfectious and chronic hepatitis 1 , hepatitis 4, 1 .Iridaceae^Belamcanda chinensis DC.flu g, hepatitis f, g, (4,7)used by Dr. He Shixiu for flu,sample collected from Dr. He.**^BUPL* *^PLPLPL**^PL**^PLLamiaceae(Labiatae)Coelogyne corymbosa Lindl.flu 1, 4 .Acrocephalus indicus Briq.cold d.Elsholtzia blanda (Benth.) Benth.flu 1,4 , hepatitis 4 .Elsholtzia bodneri Vant.hepatitis 1 .Elsholtzia ciliata (Thunb.) Hylanderor E. rugulosa Hemsl.cold 1, 4, f, flu 1, 4 .used by Dr. He Shixiu for fluElsholtzia densa Briq.flu 4 .used by Dr. He Shixiu for flu,sample collected from Dr. He.Elsholtzia penduliflora W .W . Sim^ LFor E. flava (Benth.) Benth.jaundice hepatitis 1, , flu 1, 4 , cold 4, h.22Plant Family^Species^ Part Used #Lamiaceae^Perilla frutescens (L.) Britton^**^PLcold 6, 8, f, flu 3 , (4).used for cold by Dr. He Shixiu,sample collected from Dr. He.Rabdosia phyllostachys (Diels) Hara^**^PLcold f, other Rabdosia spp. used for cold 4, f.used by Dr, He Shixiu for cold,sample collected from Dr. He.Scutellaria orthocalyx Hand.-Mazz.^**^PLhepatitis 1 , (4,0,other Scutellaria spp. used for cold.Stachys kouyangensis (Vaniot) Dunn^**^PLacute and chronic hepatitis 1 , (4).used by Dr. He Zhegao for hepatitisLiliaceae^Polygonatum kingianum Coll. et Hemsl.^PLchronic hepatitis 1 , (4).Tupistra chinensis Baker^ RHcold 1 , flu 1 , (4). +Loganiaceae^Buddleja off cinalis Maxim^ PL,FLhepatitis a , c, supports liver 4, 7, 8 .Malvaceae^Sida szechuanensis Matuda^ PLhepatitis 4, b, (1)Urena lobata L.^ LF,RTflu 1,5 .Menispermaceae^Sinomenium acutium (Thunb.) Rehd. et Wils.^LFcold 1 , bronchial infections 1 , (4).23Plant Family^Species^ Part Used #Myrsinaceae^Ardisia mammilata Hance^ PLhepatitis 1, 4, a.Embelia sessiliflora Kurz^**^RTor E. ribes Burm. f.hepatitis 1, 2, b , f, (4)Maesa indica Wall.^ *^PLhepatitis 4, a.Oleaceae^Carissa spinarum A. DC.^ PLacute and chronic hepatitis 1 .Oxalidaceae^Oxalis corniculata L.^**^PLcold 1 , jaundice hepatitis 1, 4, (7,j,g).Passifloraceae^Adenia cardiophylla (Mast.) Engl.^ PLinfectious hepatitis 1 . +Passiflora wilsonii Hemsl.^ PLhepatitis 1, a , c , g.Plumbaginaceae^Plumbago indica L.^ *^PLhepatitis b, (4).collected from medicinal plant garden inDai village, Menglun, Xishuangbanna.Poaceae^Cymbopogon distans (DC.) Stapf.^ PL(Graminae)hepatitis 1 , (3).Phyllostachys sp. (gold bamboo)^**^PLhepatitis b.Polypodiaceae^Stenoloma chusanum (L.) Ching^**^PLcold 1 , flu 4 , infectious hepatitis 1used by Dr. He Shixiu for cold,sample collected from Dr. He.Plant Family Species Part Used #Primulaceae Lysimachia christinae Hancejaundice hepatitis 1, 4, bronchial infection 1 .PLLysimachia clethroides Dubyjaundice hepatitis 1 , (4)).PLRubiaceae Hedyotis uncinella Hook. et Arn.or H. capitata Wall.prevent and cure jaundice hepatitis 1,hepatitis 1, f, g.** PLMorinda angustifolia Roxb.hepatitis a.* RTPaederia scandens (Lour.) Merr.flu 1 , bronchial infection 1 , cold 8 , (4).* PLRutaceae Boenninghausenia sesilicarpa Levl.or B. albiflora (Hk.) Meissn.cold f, hepatitis 1 , flu g, (a).** PLClausenia excavata Burm. f.flu^1, 4, b .* LFEvodia lepta (Spr.) Merr.prevent and cure flu 1 , hepatitis 1, 4 , (8).* PLSapindaceae Sapindus rarak DC.hepatitis 1, b.* LFSantalaceae Thesium himalense Roylecold 1 , bronchial infection 1 .PLSaururaceae Houttuynia cordata Thunb. ** PLcold g, flu 4, f, jaundice hepatitis 1 , (6,7,8,9)24Plant Family Species Part Used #Scrophulariaceae Siphonostegia chinensis Benth.jaundice hepatitis f, hepatitis 4 , (3,7,8)used by Dr. He Zhegao for hepatitis.** PLSolanaceae Solanum spirale Roxb.hepatitis a , b, cold 4, (i).PLVerbenaceae Verbena officinalis L.cold f, flu 3, 4 , hepatitis f, (1,5,7,8,g).** PL# Part used: BK, bark; BR, branch; BU, bulb; FL, flower; FR, fruit; LF, leaf;PL, whole plant; RH, rhizome; RT, root; SD, seed.* * Assayed for antiviral activity.* Sample collected but left in Yunnan.( ) Species listed in document, but not cited for use in the treatments of cold, flu, orhepatitis.+ Plant species had a more specific range of treatments.25Table 1.2Phylogenetic Distribution of Plants Selected as Candidates for Antiviral Screening(shown by order and family with number of species)Magnoliopsida (Dicots)Magnoliidae^Rosidae^ Asteridaesubclass total 3^subcalss total^20^subclass total^3426RanunculalesMenispermaceae 1Berberidaceae^1PiperalesSaururaceae^1Dillenidaesubclass total^15CapparalesBrassicaceae^1(Crucifereae)DillenialesDilleniaceae^1EbenalesEbenaceae^1MalvalesMalvaceae^2ViolalesPassifloraceae^2Primulale4Myrsinaceae^3Prim ulace ae^2ThealesHypericaceae^2ApialesApiaceae^1(Umbelliferae)Araliaceae^1CornalesCornaceae^2EuphorbialesEuphorbiaceae 4FabalesCaesalpinaceae 1Fab aceae^2(Leguminosae)GeranialesOxlalidaceae^1MyrtalesCombretaceae^1Plum b aginalesPlumbaginaceae 1RutalesRutaceae^4SantalalesS antalaceae^1SapindalesSapindaceae^1AsteralesAsteraceae^6(Compositae)DipsacalesDipsacaeae^1GentianalesApocynaceae^1Asclepiadeaceae 1Gentianaceae^2Loganiaceae^1LamialesLamiaceae^12(Labiatae)Verbenaceae^1OlealesOleaceae^1RubialesRubiaceae^4ScrophularialesScrophulariaceae 1SolanalesConvolvulaceae 1Solanaceae^1Liliopsida (Monocots)Arecidae^ Commelinidae^LiliidaeArecales Cyperales LilialesArecaceae Poaceae^2^Iridaceae^2(Palmae)^(Graminae) Liliacae 2Aral esAraceaeCycadophyta (gymnosperm)^Pterophyta (fern)Cycadales^ FilicalesCycadaceae^1 Polypodiaceae^1DiscussionThe use of an ethnopharmacological approach to screen for bioactive compoundsinvolves extensive surveying of information. The sources often include local publications andoral traditions in many parts of the world. Research on the medicines in turn generates newinformation. The need to manage this quantity of information for centralized access and cross-referencing has led to the development of large-scale computer databases (Loub, et al., 1985).The data on traditional Chinese medicine are assembled at the Chinese Medical MaterialResearch Centre at the Chinese University of Hong Kong. The Natural Products Alert(NAPRALERT) system at the University of Illinois, Chicago, aims to compile all chemical,pharmacological and ethno-medical data on natural products (Farnsworth, 1990).Ethnobotanical knowledge is a valuable resource that should be preserved and studied and it isimportant to have the information organized in order to facilitate further research.The 82 plant species that were selected as potential candidates for antiviral testing arewidely distributed in the taxonomic organization of higher plants. The 40 angiosperm familiesoccur in 31 orders following the taxonomic classification scheme of Cronquist (1981).Seventy-two species are in the class Magnoliopsida (dicotyledons), with 34 species in the mostrepresented subclass Asteridae. Eight species are in the class Liliopsida (monocotyledons). Inthe 31 species that were assayed for activity, 22 plant families were represented. Theyincluded a pteridophyte, a gymnosperm cycad, two monocot families (Poaceae, Iridaceae) and18 dicot families (Apiaceae, Apocynaceae, Asteraceae, Berberidaceae, Combretaceae,Convolvulaceae, Ebenaceae, Euphorbiaceae, Fabaceae, Gentianaceae, Lamiaceae, Oxalidaceae,Myrsinaceae, Rubiaceae, Rutaceae, Saururaceae, Scrophulariaceae, Verbenaceae). The speciesalso show a range of geographical distributions, from cosmopolitan (i.e. Oxalis comiculata)to regionally limited (i.e. Cycas siamensis ). The family represented most frequently is themint family Lamiaceae (Labiatae), from which six species were assayed. The wide distribution27of the plants selected reflects the fact that the pharmacopoeia surveyed includes all major plantgroups and most angiosperm families. Six of the families in this sampling were from familiesthat have more than 100 species in the Chinese medical flora (Xiao, 1983):Apiaceae, Fab aceae ,Asteraceae, Lamiaceae,Euphorbiaceae, Rubiaceae.The selection criteria used were of an ethnopharmacological rather than taxonomical nature, andit appears that a wide range of plants were used in the treatment of the viral diseases: the cold,influenza, and hepatitis.The composition of the sampling was nevertheless influenced by two factors. Plantswere screened as candidates for collection from a survey of traditional medicine used in thetreatment of specific diseases. Actual collections were made on the basis of availability ofquantities for bioactivity assays, and there may therefore have been an inherent bias against thecollection of rare or of very small plants. The frequent usage of Lamiaceae species (of the mintfamily) as medicinal plants could contribute to their high occurrence in such a survey-basedselection. It is possible that mint plants may have been also used more frequently to treat oneof the diseases used in the survey. In folk use, mints seem to have a history of uses in thetreatment of colds generally associated with the aromatic nature of the herbs having an effect onthe respiratory system. Since this survey used the indication for treatment of colds as aselection criterium, the taxonomic distribution of the plants selected could be influenced by anytendencies for a plant family to be used more commonly in folk medicine. The preponderanceof certain families of plants in traditional medicine is probably based on observations ofparticular properties of the plants. These properties can in turn be attributed to characteristics inthe chemistry of the plant family (Schultes and Swain, 1976). Swain (1972) has discussedhow the origin of modern taxonomy can be traced to the practice of medieval medicine.28In this survey, a plant was considered for antiviral screening if the treatment of one ofthe viral diseases was one of its uses. Most of the plants surveyed were not used only forthese diseases. It is difficult to make a direct link between the traditional uses of plants and thespecific viruses that are now known to cause the diseases they are used to treat. The records ofhepatitis treatment are variable, with several different types of inflammation of the liver beingdistinguished. The term for 'hepatitis' in Chinese is 'liver inflammation'. This is notinconsistent with the western diagnosis of hepatitis which also acknowledges several types ofaffliction of the liver. There are at least five different types of viruses causing hepatitis and thecausative agent is not distinguishable by symptoms or even biochemical tests (Hoofnagle andDi Bisceglie, 1990). All uses of plants for treating liver inflammation and jaundice were takenas an indication of treatment for hepatitis. Han et al. (1988) conducted a survey of theeffectiveness against viral hepatitis of 97 Chinese medicines. The natural materials selected aspotential candidates includes species of nine plant genera (Elsholtzia, Halenia, Houttuynia,Lysimachia, Morinda, Polygonatuni, Scutellaria, Swertia, Verbena). Species of these generahave all been cited for the treatment of hepatitis in Yunnan. Swertia spp. and Halenia spp. arereported to show effectiveness in clinical trials. Common colds are considered to be mostfrequently caused by rhinoviruses, but coronaviruses and enteroviruses may cause similarsymptoms; just as flu-like syndromes can be caused by influenza and parainfluenza, as well asother respiratory tract viruses (Lycke, 1983). Plants used solely for treatments of symptomsrelating to cold and flu afflictions, such as cough suppression or reduction of phlegm, wererejected for the screen, but those used in the treatment of respiratory tract infections wereconsidered. On the basis that the use of a plant for treatment of cold, flu or hepatitis was anindication for potential antiviral activity, 69 plant genera occurring in Yunnan were identified ascandidates for screening.Representatives of 48 plant genera were collected during two months in Yunnan.Eighteen of the species collected were not assayed for antiviral activity. This was due to29unforeseen limitations imposed by a contract which was drawn up between the EthnobotanyLaboratory at the Kunming Institute of Botany and another institution preventing furtherresearch collaborations between the Laboratory and me on specified plant species for a periodof time. This unexpected restriction is a reminder that factors of commercial or proprietaryinterest may arise in the course of research on the traditional medicinal knowledge and thenatural resources of any culture or countries. It is integral to the ethnobotanical researchapproach to develop and maintain good collaborative relationships with the people whoseknowledge and resources are involved in the research, and this unexpected development wasaccepted.30Chapter I. ReferencesCronquist, A. (1981) An Integrated System of Classification of Flowering Plants. ColumbiaUniversity Press, New York, 1262 pp.Duke, J.A. and Ayensu, E.S. (1985) Medicinal Plants of China Vol. 1. ReferencePublication, Algonac, Michigan, p. 40.Farnsworth, N.R. (1990) Ethnopharmacology and drug development. In: BioactiveCompounds from Plants. Wiley, Chichester (Ciba Found. Symp. 154), pp. 2-11.Grey-Wilson, C. (1988) Journey to the Jade Dragon Snow Mountains, Yunnan I. Quart. Bull.of the Alpine Garden Society 56 (1), March 1988, No. 231, 16-33.Han, D.W., Xu, R.L. and Yeung, S.C. (1988) Abstracts of Chinese Medicines, 2 (1), 105-134.Hoofnagle, J.H. and Di Bisceglie A.M. (1990) Antiviral therapy of viral hepatitis. In: G.J.Galasso, R.J. Whitley and T.C. Merigan (eds.) Antiviral Agents and Viral Diseases ofMan, 3rd Edition, Raven Press, New York, pp. 415-459.Huang, L. (1984) Drug research based on the leads of the Chinese traditional medicine. In: P.Krogsgaard-Larsen et al. (eds.), Natural Products and Drug Development, AlfredBenzon Symposium 20, Munksgaard, Copenhagen, pp. 94-105.Huang, S.Y. (1984) Introduction to Chinese Medicine. [in Chinese], Ba De EducationalPublications, Taipei, 150 pp.Kunming Institute of Botany (1978) Chinese Medicinal Plants of Yunnan, [in Chinese],Tiangjing People's Publishing, Tiangjing, 535 pp.Kunming Reserve Unit Health Division (1970) Chinese Medicinal Plants of Yunnan. [inChinese], Tiangjing People's Publishing, Tiangjing, 777 pp.Lancaster, R. (1981) Dominant woody vegetation at expedition camps on Cangshan Range.Report of the Sino-British Botanical Expedition to China.Li, S. (1979) Ben Cao Gang Mu. compiled in 1578. [in Chinese], People's HealthPublications, Chengdu, 1432 pp.Loub, W.D., Farnsworth, N.R., Soejarto, D.D. and Quinn, M.L. (1985) NAPRALERT:computer handling of natural product research data. J. Chem. Inf. Comput. Sci. 25,99-103.Lycke, E. (1983) Viral syndromes. In: E. Lycke and E. Norrby (eds.) Textbook of MedicalVirology. Butterworths, London, pp.341-350.Pei, S. (1988) The status of ethnobotany in China. Paper presented to the First InternationalCongress of Ethnobotany, Belem, Brazil, July 19-25, 1988.31Schultes, R.E. and Swain, T. (1976) The plant kingdom: a virgin field for new biodynamicconstituents. In: N.J. Fina (ed.) The Recent Chemistry of Natural Products, IncludingTobacco. (Proceedings of the Second Phillip Morris Science Symposium), New York,pp. 133-171.Swain, T. (1972) The significance of comparative phytochemistry in medical botany. In: T.Swain (ed.) Plants in the Development of Modern Medicine. Harvard UniversityPress, Cambridge.Xiao, P. (1983) Recent developments on medicinal plants in China. J. Ethnopharm. 7, 95-109.Yunnan Institute of Tropical Botany (1984) List of Plants in Xishuangbanna. [in Chinese],Yunnan Minority Publication, Yunnan, 248 pp.Yunnan Institute of Medicine Inspection (1983) List of Yunnan Minority Tribe Medicines. [inChinese], Yunnan Minority Publications, Yunnan, 112 pp.Zhang, A. (1983) Rhododendrons of the Nujiang (Salween) Valley of Yunnan Province,People's Republic of China. Notes RBG Edinb. 41(1), 141-164.Zou, S. (1988) The vulnerable and endangered plants of Xishuangbanna Prefecture, YunnanProvince, China. Arnoldia, 48(2), 1-7.32Chapter II.Screening for Antiviral ActivityIntroductionProgress in the discovery of chemical agents for the treatment of viral diseases has insome cases reached the stages of clinical use and trials (Becker, 1984; Allison, 1986). One aimof antiviral research is toward the development of compounds that can inhibit viral infectionselectively without being harmful to the virus host. A few mostly synthetic compounds havebeen approved for therapy, and several natural products have been found to be promising leadsas antiviral agents and are under further study (Hudson, 1990, Vlietinck and Vanden Berghe,1991). The studies of antiviral extracts and compounds derived from plants are summarized byChe (1991). About 4000 plants are estimated to have been screened for antiviral effects, andover 450 species that have shown in vitro activity are listed. Zheng (1988) tested 472 Chinesemedicinal herbs for activity against herpes simplex virus type 1 and 31 (7.2%) of the alcoholicextracts of these species was found to be active in in vitro tests. The pandemic threat of theretrovirus-linked acquired immunodeficiency syndrome (AIDS) has given impetus to efforts tofind antiviral agents that inhibit the human immunodeficiency virus (HIV), and manycompounds have been tested for this effect (De Clercq, 1987). In a screening of 27 Chinesemedicinal herbs, extracts of Viola yedoensis Maxim showed inhibition of HIV (Chang andYeung, 1988). In addition, several compounds of different chemical classes from medicinalplants have shown anti-retroviral activity. These include: sulfated polysaccharides from theChinese medicinal plant Prunella vulgaris L. (Tabba, et al.,1989), the naphthabianthronehypericin from Hypericum spp. (Meruelo et al., 1988), the indolizidine alkaloidcasatanospermine from the seed of the Australian tree Castanospermum australe A Cunn. etFraser (Walker et al., 1987), and the protein trichosanthin from the tuber of another Chinesemedicinal plant Trichosanthes kirilowii Maxim (McGrath et al., 1989). Methoxyflavones are33active against picornavirus. These compounds were isolated from both the Chinese medicinalplant Agastache rugulosa 0. Kuntze (Ishitsuka et al., 1982) and the African medicinal plantEuphorbia grantii Oliver (Van Hoof et al., 1984). These examples of antiviral plantcompounds demonstrate the effectiveness of screening medicinal plants in the search forantiviral agents. An overview of the process of developing antiviral agents from plants waspresented by Vanden Berghe et al. (1985).The virus is an obligate intracellular parasite. It differs from other parasitic organismsin that it is dependent on the host at the genetic level by requiring the use of cellular molecularmechanisms in its replication. It is basically composed of nucleic acid genetic material enclosedwithin a protein nucleocapsid. The nucleic acid can be a single or double strand, consisting ofribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The protein capsid is eithericosahedral or helical in shape but a few, such as the poxvirus, have more complex symmetry(Choppin, 1986). The capsid can be nonenveloped or membrane enveloped. Thenucleocapsid is encased by a lipid bilayer membrane in about half of known animal viruses(Hudson, 1990). The enveloped virus has been defined by Matthews (1983) as "having abounding lipoprotein bilayer membrane necessary for infectivity in normal conditions".Virus taxonomy is complicated and the grouping of a virus often reflects its host and/orassociated disease. Viruses are often very host specific. Most generally, viruses can becharacterized by nucleic acid and particle types (Norrby, 1983). Changes in classification haveevolved with the advancement of knowledge and technology that have led to the examination ofrelationships according to antigenicity (van Regenmortel, 1982), replication strategies (Hersheyand Taylor, 1986) and phylogeny (Goldbach, 1990). Matthews (1983) reviewed the historyand status of virus taxonomy. Viruses appear to have been very successful in exploiting allforms of life, being ubiquitous in animals, plants and bacteria. Viruses have developed manydifferent molecular strategies of replication and many mutate rapidly. They can persist within34the host and manifest chronic symptoms or remain latent until an episode of reactivation.Viruses have also adapted to numerous vectors of transmission between hosts. All thesefeatures about viruses contribute to the complexity of the task of controlling the spread ofdisease-causing viruses.The different possible sites of action for antiviral agents are related to the differentstages of viral replication. The sequence of events have been comprehensively described byMitchell (1973). Briefly, virus infection and reproduction include the following stages:1. adsorption to and penetration of the host cell,2. uncoating of the viral particle,3. transcription and translation of viral nucleic acids,4. replication of genome,5. assembly and release of progeny virus.The antiviral agent could target any of these stages. As viral replication depends on cellularmolecular mechanisms to some extent, the ideal antiviral agent would have an irreversible effectonly on a virus-specific component in one of these stages (Sim, 1990). The majority ofantiviral drugs in use are nucleoside analogs that interfere in viral replication by disruptingnucleic acid polymerization, but they have toxic side effects due to the similarity between theviral and the normal cellular process (Montgomery, 1989). The chemical interferences at thesedifferent stages will be further discussed in Chapter V which concerns the nature of antiviralaction.For the evaluation of the antiviral potentials of an extract or a chemical, in vitroantiviral assays using animal cell culture systems have become established procedures and arereviewed by Hu and Hsiung (1989). Tests for antiviral effects are based on measurementsindicative of virus replication in a suitable host cell system. Antiviral activity can be seen as theinhibition of cytopathic effects (CPE) caused by the virus in the presence of the agent tested.35This method is applicable to viruses that cause a visible change in appearance of infected cellsand is useful for testing a range of concentrations. The effective concentration is determined bythe absence of visible CPE. A method using a colored reagent has been developed for the invitro CPE inhibition assay of HIV (Weislow et al., 1989). This allows for the quantificationof drug effects with colorimetric techniques. With viruses that form plaques on cellmonolayers indicating units of infection, the reduction of plaque numbers in a treated versus acontrol cell layer gives a quantitative assay of antiviral activity. This method can be used toobtain a dose-response curve by comparing the plaque reductions with different concentrationsof compound tested (Hudson et al., 1991). Similarly, in the virus yield reduction assay, virusinfected cells are treated with a range of concentrations of the agent tested. The virus yields arethen assayed after a suitable incubation time (Boyd et al., 1987). Hu and Hsiung (1988)compared plaque reduction and virus yield reduction assays and found the results to be similar.Not all viruses form plaques or induce visible CPE. Other methods have been used thatmonitor viral replication by measuring a virus-specific effect or product. Reverse transcriptaseis a RNA-directed DNA polymerase that is virus coded and necessary in the replication ofseveral viruses (Doolittle et al., 1989). The inhibition of reverse transcriptase activity can beused as an indication of an antiviral effect. This method has been used in studies with HIV(Nakashima et al., 1987; Nishizawa et al., 1989) and RNA tumor viruses (Sethi, 1985). Viralantigens in cell cultures can be detected with enzyme-linked immunosorbent assays (ELISA) orimmunofluorescence assays (Rabalais et al., 1987; Farber et al., 1987). Effect on viralreplication can be detected with the quantification of viral nucleic acids using molecular biologytechniques such as RNA-DNA hybridization (Lin et al., 1987). Virus particles can beobserved under an electron microscope. Absence of virions or defective virions can alsodemonstrate antiviral activity (Fong et al., 1987). A tabular summary of the differencesbetween applicability of in vitro antiviral assays is found in Vlietinck and Vanden Berghe(1991).36The biological activity of many plant constituents can be dependent on, or enhanced by,simultaneous irradiation with light (Towers, 1980). Antiviral plant photo-sensitizers,particularly those activated by long wavelength ultraviolet (UVA, 320-400 nm) radiation, havebeen reviewed by Hudson (1989). To test for the possibility that the activity of some plantextracts might be light-mediated, the bioassays were done in the presence and absence of UVAradiation.The crude extracts of 31 medicinal plant species from Yunnan, China, were assayed forantiviral activity. These species are distinguished on the list of plants selected as potentialcandidates for testing which is in Table 1.1 of Chapter I. They are also listed separately inAppendix I. The extract determined to be the most active was also tested for antibacterial andantifungal activities.Material and MethodsCell cultureTwo monolayer-forming mammalian cell lines from the American Type CultureCollection were used for the bioassays. The 3T3-L1 is a mouse fibroblast cell line and Vero isan African green monkey kidney cell line. The cells were grown in a 5% carbon dioxideatmosphere at 37 °C, in Dulbecco's Modified Eagle A Medium (DMEM) with 10% fetal bovineserum (Gibco) and 25 pg/ml gentamicin sulfate (Sigma).37Cytotoxicity AssaysCell layers of 3T3-L1 were grown in 96 well microtiter plates (Falcon 3072).Cytotoxicity of plant extracts was first tested by exposure of the cells to dilutions of the extractsin the cell culture medium. Two treatments were done: with the extracts added when the cellswere still in suspension prior to monolayer formation, and when the cells had formed confluentmonolayers. The highest concentration of extract tested was the equivalent of 100 mg ofstarting plant material per ml, from the ethanol soluble portion of crude ethanolic extractresidues. Two-fold serial dilutions were made from this concentration and added to the cells.The treated cells were incubated and observed for cytopathic effects.Assay VirusesTwo animal viruses were used to screen for antiviral activity. Sindbis (SINV) is asingle-strand RNA virus of the Togaviridae, size - 40 nm. Murine cytomegalovirus (MCMV)is a double-strand DNA virus of the Herpesviridae, size - 200 nm. Both of these aremembrane-enveloped viruses. Some species that were active in the screen were assayedagainst a nonenveloped virus with human polio virus 1. Polio is a single-strand RNA virus ofthe Picornaviridae, size - 25 nm. All three viruses have icosahedral capsid symmetry.Antiviral Screening AssaysTwo-fold serial dilutions of the plant extracts were tested starting with non-cytotoxicconcentrations. The infectivities of the two viruses were assayed qualitatively throughmicroscopic observations of characteristic viral cytopathic effects (CPE). Controls of cellsonly and untreated virus infections were done with each assay for comparison. The viraleffects radiate from centers of infection. The CPE of SINV is seen as shrunken and highly38refractive cells, and MCMV causes cell swelling. Antiviral activity was determined by theabsence of CPE in a treatment. The minimum active concentration was the lowestconcentration of extract at which the absence of CPE was observed. The known UVA-enhanced antiviral plant compound oc—terthienyl (Hudson et al., 1986) was used as an antiviralcontrol.In a comprehensive assay , three separate segments of the viral life cycle were exposedto the plant extracts successively. The cell preparations were incubated with the extractdilutions in culture medium for 24 hrs during monolayer formation. The viruses, at aconcentration of 1000 plaque forming units (pfu) per ml, were exposed to the same extractdilutions in phosphate buffered saline (PBS) at 4 ° C for 30 min. The cell layers were thenexposed to the virus/extract mixture for 1 hr of adsorption time at 37 ° C. After removal of thevirus mixture, the cells were incubated in medium with the same extract dilutions. Two sets ofcontrol cells were subject to the same manipulations: one set without virus or the plant extracts(cell control), one set exposed to the virus but without plant extracts (virus control). Controlassays were done with dilutions of all the solvents used in the addition of the extracts todetermine the concentrations below which there were no solvent effects. The plant extractswere usually prepared with methanol or ethanol.In the multiple-treatment assay, 16 plant extracts showed inhibition of viral infection.These extracts were assayed again following the same procedure but with three parallelpreparations of cells. Instead of one set of cells being subject to three successive extracttreatments, each set was exposed to only one of the treatments. Each treatment was done inparallel with and without exposure to UVA radiation The time of extract treatment for each setis shown in the following scheme:3940^Time of Extract TreatmentPre-infection^I Infection Time^I Post -InfectionSet 1Cells only for 24 hrsprior to addition ofvirusSet 2Virus only for 30 minand with cells during1 hr of infection timeSet 3Cells only post-infection afterremoval of virusFurther assays were repeated with extracts of eight species. The respective extracts inethanol were filtered (Whatman paper No. 1) to remove undissolved crude residues,evaporated to dryness and the residues taken up in 100% methanol back to the same volumes.The methanol fractions were assayed following the same procedure, with extract treatment ofthe virus suspension and the cells during virus adsorption.Plaque Reduction AssaysCell monolayers were formed in petri dishes (Corning 25010, 60mm diameter, forSINV and polio assays; Corning 25000 , 35 mm diameter, for MCMV assays). The cells wereexposed to the extract / virus mixture for 1 hr of adsorption time, with 1000 pfu of virus pertreatment. After removal of the virus mixture, the cells were overlain with medium in 0.5%agarose and incubated until viral plaques were visible. The cells were sometimes fixed with3.7% formaldehyde in PBS and stained to facilitate plaque counting (crystal violet for SINVand polio, methylene blue for MCMV). Cell and virus controls were done with each assay.Plaque reduction is calculated as a percentage from the number of plaques compared to thevirus-only control. An example of the plaque reduction assay is shown in Figure 2.1.41Figure 2.1: Plaque Reduction Assay of Antiviral Activity.UVA IrradiationThe UVA radiation was provided in a temperature-controlled chamber (Environ-Shaker3597, Lab-Line Instruments) by a bank of 6 Philips F20T12/BLB light tubes, with peakemission at 350 nm and an incident energy of 270 1.1W/cm2 . An extract was considered toshow enhancement of activity by UVA radiation when antiviral effects were observed at lowerconcentrations of the extract, compared to the parallel culture kept in the dark.Antibiotic assaysBacterial and fungal cultures in liquid media were spread on nutrient agar plates(Muehler-Hinton medium for bacteria, yeast nitrogen base medium for Saccharomyces). Plantextracts were pipetted in 20 p,1 aliquots on sterile filter paper disks 6 mm in diameter(Schleicher & Schuell 740-E). The plates were incubated for 24 hrs at 37 ° C. Parallel plateswere incubated in the dark and with exposure to UVA radiation. Antibiotic effects were seenas clear zones of culture inhibition surrounding the paper disks.ResultsAntiviral Activity ScreeningThe highest concentration used for each extract was determined by the cytotoxicityassays. Extract concentrations used for the antiviral assays were in the range equivalent to 301.1g/m1 to 1.0 mg/ml of original plant material. The crude extracts of 31 plant species wereassayed and 16 of them showed antiviral activity. The results are shown in Table 2.1. Theconcentrations shown were calculated from weight of dry plant material extracted.42Table 2.1Antiviral Activity of Crude Extracts of Yunnan Medicinal PlantsAgainst Murine Cytomegalovirus (MCMV) and Sindbis Virus (SINV)Plant species^Minimum Active Concentration (tg/m1)# UVAenhancementlisted by family MCMV^SINVApiaceaeCentella asiatica (L.) Urban 65AsteraceaeBidens pilosa L. 125 65CombretaceaeQuisqualis indica L. 125ConvolvulaceaeDichondra repens Forst. 125 125CycadaceaeCycas siamensis Miq. 160EuphorbiaceaeEuphorbia prolifera * 65Ehrenb. ex. Boiss.IridaceaeBelamcanda chinensis DC. 125LamiaceaeAcrocephalus indicus Briq. * 65 65Elsholtzia densa Briq. 125Elsholtzia ciliata (Thunb.) Hylander * 65 30 +Rabdosia phyllostachys (Diels) Hara 125Stachys kouyangensis (Vaniot) Dunn * 65 +MyrsinaceaeEmbelia sessiliflora Kurz * 30 30RutaceaeBoenninghausenia sessilicarpa Levl. * 80 +ScrophulariaceaeSiphonostegia chinensis Benth. * 40VerbenaceaeVerbena officinalis L. * 65 30* further assay results shown in Table 2.2. # based on dry wt. of plant material extracted43The 16 active extracts were assayed again when the cell cultures were exposed toextract treatments separately at the pre-infection, virus / infection, and post-infection stages.The antiviral activity in all cases occurred only upon treatment of the virus and of the cellsduring infection time. Eight extracts of higher activity were selected for further assays. Theresults from the assays of the filtered methanolic fractions are shown in Table 2.2.Activity of Elsholtzia ciliata and other Elsholtzia speciesThe highest activity in the CPE inhibition assay of methanolic fractions was shown byElsholtzia ciliata with the minimum active concentration of 2 µg/ml. The assay of a separatepreparation of crude extract was against Sindbis virus at 0.3 pg/ml with exposure of the virus /extract to UVA. The UVA light enhancement of activity of E. ciliata occurred even if only thevirus / extract mixture was exposed to light and the virus adsorption period on the cellmonolayer was in the dark. One sample of dried plant material obtained from the EthnobotanyLaboratory of KIB was not active. The sample was much more woody than the othercollections. Crude extract and solvent fractions of E. ciliata had no activity against polio virusin plaque reduction assays. Alcoholic crude extracts of E. splendens Nakai ex F. Maek. (fromKunming) and E. blanda (Benth.) Benth. (from Nepal) were active against SINV in plaquereduction assays at a concentration equivalent to 2 mg/ml of dry plant material, with UVA light.A sample of the essential oil of E. ciliata previously distilled by the Kunming Institute ofBotany showed a narrow range of marginal activity in plaque reduction assays between itscytotoxic and inactive concentrations (1:1000 to 1:1500 dilutions).Antibiotic assaysThe results of the antibiotic assay of the E. ciliata crude extract are shown in Table 2.3.The extract concentration was the equivalent of 40 pg of dry plant material.44Table 2.2Minimum Active Concentrations of Methanolic FractionsAgainst Murine Cytomegalovirus (MCMV) and Sindbis Virus (SINV)Minimum Active Concentrations (lig/m1)# LightMediationPlant species^MCMV^SINVAcrocephalus indicus 125 125Boenninghausenia sessilicarpa 65 UVA500 darkElsholtzia ciliata 2 2 UVA65 65 darkEmbelia sessiliflora 15 4Euphorbia prolifera 30Siphonostegia chinensis 65Stachys kouyangensis 65 UVAlmg darkVerbena officinalis 30 30# concentrations calculated from weight of dry plant material extracted45Table 2.3Antibiotic Activity of the Crude Extract of Elsholtzia ciliataDiameter of Inhibition Zone (mm)*46Micro-organismStaphylococcus aureussensitive strainStaphylococcus aureusresistant strainStaphylococcus aureusmethicillin resistantEscherichia coli DC2Pseudomonas sp.Saccharomyces cerevisiaemethanol controlgent. control (E. coli)gent. control (S. aureus sens.)fung. control (S. cerevesiae )UVA Dark10 712 7(--) (--)6.5 6.5(--) (--)10 (--)(--) (--)16 1425 2512 12* diameter of assay disk = 6 mm^(--) = no inhibition zonegent. = antibiotic gentamicin sulfate (Sigma), 10 tg/disk.fung. = antifungal fungizone (Gibco), 10 tg/disk.Extract concentration = 40 pg of dry plant material.Incubation 24 hrs. in UVA or in dark at 37°C.DiscussionThe screening method used here comprised a general antiviral screening approach ratherthan one designed to assay specifically for the viruses related to the illnesses that the plantswere used to treat. The design of the screening program included a feasibility aspect.Compared to in vivo assays, in vitro bioassays are a cost and effort effective approach toantiviral screening, although there are differences in the ease and viability of assaying differentviruses, such that in practicality not all viruses of chemotherapeutic interest for investigation areamenable to this system. Vanden Berghe and Vlietinck (1991) described the composition of abattery of six viruses that represent a range of virus morphology and diseases that they used inscreening of plant extracts (adeno-, measles, Coxsackie, herpes simplex, polio-, Semlikiforest). The last four are from the same families as the three used in this study.The two viruses used in the general screening assays represent two major divisions invirus classification based on the nature of the nucleic acids: RNA vs. DNA, and single-strandvs. double-strand (SINV = ssRNA, MCMV = dsDNA), but these are both membraneenveloped. The third virus used in further assays is a nonenveloped virus (polio = ss RNA).The characteristics of SINV will be discussed in more detail in Chapter V in the examination ofantiviral mechanisms. Regarding the viruses that cause the diseases considered in the plantselection: rhinovirus (cold) is also a picornavirus and influenza viruses are ss RNAorthomyxoviruses, with membrane envelopes. Not all of the different hepatitis viruses havebeen fully characterized but they include the nonenveloped ss RNA hepatitis A picornavirus.Hepatitis B is a dsDNA hepadnavirus. It is not membrane enveloped but has instead anenvelope consisting of the viral coat protein, lipids and carbohydrates (Summer et al., 1983).By comparing the overlap in virus grouping using more than one morphological feature,Matthews (1983) has proposed that using the presence or absence of the membrane envelopewould be a more "informative and predictive" primary division of the virus groups than the oneusing the RNA vs. DNA criterion. From the perspective of studying the bioactive nature of47compounds, the presence or absence of a membrane envelope would also be an importantfeature to consider because the envelope could be a different site of action. The extract ofElsholtzia ciliata, which was active against the two membrane bound viruses, was not activeagainst the nonenveloped polio virus. This suggests that the antiviral action of this particularextract could involve the virus membrane.Six of the extracts active against SINV were also active against MCMV. None wereactive against MCMV only. In general, activity was observed at lower extract concentrationsagainst SINV than against MCMV. Besides the differences in the nature of their nucleic acids,MCMV is also a much larger virus in size and genome. A simple explanation for the differencein sensitivity could be that it was due to the size difference, especially possible if the virusmembrane was the site of antiviral action.The antiviral activity shown in these assays could have taken place at several differentstages of the viral cycle. The effective treatment included exposure of the virus first to theextract and the exposure of the cells to the same virus / extract mixture. The extract could haveinhibited viral infection by affecting the virus particle itself, by interfering with the virus-hostcell recognition and entry process, or by interfering with the early steps of intracellular viralreproduction. Antiviral activity from a pre-infection treatment would indicate an interferon-stimulatory type effect. Wachsman et al. (1987) have reported this type of effect againstSindbis virus from plant extracts of the Meliaceae. Activity from a post-infection treatmentwould suggest interference in the later steps of viral replication, or in viral progeny packagingand release.In the screening process, the active concentrations of the extracts were used more asindications of relative activity. There was slight variability in the exact minimum activeconcentrations between repeated assays. An effort was made to assay the extracts48simultaneously to minimize the variability. In the assays of the crude extracts brought backfrom Yunnan, the concentrations were calculated back to the original amount of plant materialextracted. This was done because the residues brought back from Yunnan redissolvedincompletely and to different extents amongst the extracts. Only the soluble portions weretested. To continue screening assays, these portions from eight active species were separatedfrom the remaining residues, dried and taken up in methanol to the same volume.The plant extract that showed the highest antiviral activity was from Elsholtzia ciliata(Thunb.) Hylander of the mint family Lamiaceae. In the general screening assay, the extractwas active against SINV to 3014 (of original plant sample extracted) per ml, which was thelowest concentration tested, and against MCMV to 65 [tg/ml. In the methanolic fractionsassay, E. ciliata was again the most active, showing activity against both SINV and MCMV at2 tg/ml, with UVA. In assays of larger samples obtained for purification of activecomponents, it was active against SINV at 0.3 [tg/m1 (actual concentration of extract) withexposure to UVA light. On this basis, E. ciliata was selected as the most active plant for theisolation of an active component using bioassay-guided fractionation. The activity occurredeven if only the virus / extract mixture was exposed to light and the virus adsorption period onthe cell monolayer was in the dark. This indicates that the action inhibited the ability of thevirus to penetrate and infect the cell. It is likely that the virus particle was directly inactivated.The chemistry of the purification process of the active component from E. ciliata is thesubject of the next chapter. In a follow up study of the bioactivity, Elsholtzia extract alsoshowed some UVA-enhanced antibacterial and antifungal activity. The activity was notdramatic and this aspect was not further examined. The essential oil of Elsholtzia species hasbeen reported to have antibacterial and antifungal activity (Bisht et al., 1985; Chaturvedi andSaxena, 1985). It has also been reported to have insect growth retarding activity (Bhattacharyaand Bordoloi, 1986) The oil sample assayed in this study showed only weak antiviral activity49and the purification of its constituents was not pursued. The sample that contained more shootand root material was also not active, suggesting the activity may be more abundant in the leafmaterial, but since sample quantities were always limited, leaves and twigs were not separatedin further samples.The Lamiaceae was the most highly represented of the 22 plant families screened, andfive of the six species examined showed activity. Two of the active species were from thegenus Elsholtzia (E. ciliata and E. densa), and two other Elsholtzia species acquired later (E.blanda and E. splendens ) were also active. Seven Elsholtzia species were selected aspotential candidates for this screening from the pharmacopoeia of Yunnan , which was thehighest number of species for one genus. A table of the Chinese medicinal Elsholtzia speciesis presented at the beginning of Chapter III. The active species E. splendens was not on thecandidates list (Table 1.1) because it had been not cited by its Latin name in the sources onYunnan plants. It was cited in Chinese Herbal Pharmacology (Nanjing Institute ofPharmacology, 1980) that an aqueous extract of E. splendens inhibited echovirus. Since itoccurrs in Yunnan, it probably was used there medicinally because the Chinese name for E.splendens can be used for several species, including: E. blanda, ciliata, densa and rugulosa.This common nomenclatural overlap or discrepancy becomes evident when the attempt is madeto match organisms designated by traditional names with scientific taxonomic groups.Nonetheless, the high percentage of bioactivity from the genus that was found to be most citedin this informaton survey supports the hypothesis that there is a good correlation betweenbioactivity and plant groups frequently used in traditional medicines.Three species in the antiviral screening showed UVA-enhanced activity. Elsholtziaciliata and Stachys kouyangensis are mint plants and the other was Boenninghauseniasessilicarpa of the Rutaceae. The Rutaceae is known to contain furanocoumarins which arephotosensitive compounds (Towers, 1980). There have been no reports of photosensitizers50from the Lamiaceae. The four Elsholtzia species tested all showed activity and the activity wasenhanced by UVA except for the case of E. densa . This suggests that there are interestingphytochemical questions concerning the genus Elsholtzia, as well as the family Lamiaceae,which require further investigation.Ethnopharmacological information from Yunnan was used as a guideline in theselection of plants to screen for antiviral activity. The fact that 16 out of 31 plants testeddemonstrated activity supports the validity of the approach of searching for bioactivecompounds in medicinal plants. The 16 active species are in 12 plant families. In addition,five of the active species were selected for the screening based on their documented usage bythe ethnic minority tribes of Yunnan (see citation notations in Table 1.1, Chapter I). These fivespecies are:Acrocephalus indicus Brig.,Belamcanda chinensis DC.,Quisqualis indica L.,Siphonostegia chinensis Benth.,Stachys kouyangensis. (Vaniot) Dunn.The species Belamcanda chinensis, Quisqualis indica, and Siphonostegia chinensis are alsodocumented in the general literature on traditional Chinese medicine, but they are not cited astreatment for any of the viral diseases used as the screening selection criteria. Therefore, it wasfrom the minority groups' ethnopharmacological information that these plants were selected forscreening. The fact that five of sixteen active species were selected because of the examinationof additional culturally-based information concerning the same flora, indicates the importanceof both researching and preserving ethnobotanical knowledge.51Chapter II. ReferencesAllison, F. Jr. (1986) The status of antiviral therapy. In: H. Rothschild and J.C. Cohen (eds.)Virology in Medicine. Oxford University Press, New York, pp. 181-228.Becker, Y. (1984) Current trends in the research on antiviral drugs. In: Y. Becker (ed.)Antiviral Drugs and Interferon: the Molecular Basis of Their Activity. 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CRC Press, Boca Raton, pp. 219-245.McGrath, M.S., Hwang, K.M., Caldwell, S.E., Gaston, I., Luk, K.C., Wu, P., Ng, V.L.,Crowe, S., Daniels, J., Marsh, J., Deinhart, T., Lekas, P.V., Vennari, J.C., Yeung,H.W. and Lifson, J.D. (1989) GLQ223: an inhibitor of human immunodeficiencyvirus replication in acutely and chronically infected cells of lymphocyte andmononuclear phagocyte lineage. Proc. Natl. Acad. Sci. USA 86, 2844-2848.Meruelo, D., Lavie, G., and Lavie, D. (1988) Therapeutic agents with dramatic antiretroviralactivity and little toxicity at effective doses: aromatic polycyclic diones hypericin andpseudohypericin. Proc. Natl. Acad. Sci. USA 85, 5230-5234.Mitchell, W.M. (1973) Active sites of the animal viruses: Potential sites of specificchemotherapeutic attack. In: W.D. Carter (ed.) Selective Inhibitors of Viral Functions.CRC Press, Boca Raton, pp. 51-77.Montgomery J.A. (1989) Approaches to antiviral therapy. 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(1983) The morphology of virus particles. Classification of viruses. In: E. Lyckeand E. Norrby (eds.) Textbook of Medical Virology. Butterworths, London, pp. 4-16.Rablais,G.P., Levin, M.J. and Berkowitz, E. (1987) Rapid herpes simplex virus susceptibilitytesting using an enzyme-linked immunosorbent assay performed in situ on fixed virusinfected monolayers. Antimicrob.Agents Chemother. 31, 946-948.Sethi, M. (1985) Comparison of inhibition of reverse transcriptase and antileukemic activitiesexhibited by protoberberine and benzophenanthridine alkaloids and structure-activityrelationships. Phytochem., 24 (3), 447-454.Sim, I.S. (1990) Virus replication: target functions and events for virus-specific inhibitors. In:G.J. Galasso, R.J. Whitley and T.C. Merigan (eds.) Antiviral Agents and ViralDiseases of Man, 3rd Edition, Raven Press, New York, pp. 1-47.Summer, J., Mason, W.S. and Snyder, K.L. (1983) Biology of hepatitis B viruses. In: L.R.Overby, F. Deinhardt and J. Deinhardt (eds.) Viral Hepatitis. Marcel Dekker, NewYork, pp. 45-47.Tabba, H.D., Chang, R.S., and Smith, K.M. (1989) Isolation, purification and partialcharacterization of prunellin, an anti-HIV component from aqueous extracts of Prunellavulgaris , Antiviral Res. 11, 263-274.Towers, G.H.N. (1980) Photosensitizers in plants and their photodynamic action [review].Progress in Phytochem. 6, 183-202.Van Hoof, L., Vanden Berghe, D.A., Hatfield, G.M. and Vlietinck, A.J. (1984) Plantantiviral agents; V. 3-methoxyflavones as potent inhibitors of viral-induced block ofcell synthesis. Planta Medica 50, 513-517.Vanden Berghe, D.A., Vlietinck, A.J. and Van Hoof L. (1985) Present and future status ofplant products as antiviral agents. In: A.J. Vlietinck and R.A. Dommisse (eds.)Advances in Medicinal Plant Research. Wissenschaftliche, Stuttgart, pp. 47-99.Vanden Berghe, D.A. and Vlietinck, A.J. (1991) Screening methods for antibacterial andantiviral agents from higher plants. In: K. Hostettmann (ed.) Assays for Bioactivity(Methods in Plant Biochemistry Vol. 6), Academic Press, San Diego, pp. 47-70.54Vlietinck, A.J. and Vanden Berghe, D.A. (1991) Can ethnopharmacology contribute to thedevelopment of antiviral drugs? J. Ethnopharm. 32, 141-153.van Regenmortel, M.H.V. (1982) Serology and lmmunochemistry of Plant Viruses.Academic Press, New York.Wachsman, M.B., Damonte, E.B., Coto, C.E. and de Torre, R.A. (1987) Antiviral effects ofMelia azedarach L. leaves extracts on Sindbis virus-infected cells. Antiviral Res. 8,1-12.Walker, B.D., Kowalski, M., Goh, W.C., Kozarsky, K., Krieger, M., Rosen, C.,Rohrschneider, L., Haseltine, W.A. and Sodroski, J. (1987) Inhibition of humanimmunodeficiency virus syncytium formation and virus replication by castanospermine.Proc. Natl. Acad. Sci. USA. 84, 8120-8125.Weislow, 0.S., Kiser, R., Fine, D.L., Bader, J., Shoemaker, R.H. and Boyd, M.R. (1989)New soluble-formazan assay for HIV-1 cytopathic effects: application to high-fluxscreening of synthetic and natural products for AIDS-antiviral activity. J. of Natl.Cancer Inst. 81 (8), 577-586.Zheng, M. (1988) An experimental study of antiviral action of 472 herbs on herpes simplexvirus. J. of Traditional Chinese Medicine, 8 (3), 203-206.55Figure 3.1:Illustration of Elsholtziain a Chinese herbalChapter IIIPurification and Identification of Active Component from Elsholtzia ciliataIntroductionIn the screening of 31 medicinal plants for activity against the enveloped virusesSindbis and murine cytomegalovirus in mammalian cell cultures, the crude extract from theleaves and twigs of Elsholtzia ciliata (Thunb.) Hylander of the Lamiaceae showed potentUVA-enhanced activity. This species was selected as the species to purify the activecomponent from because it showed the highest activity amongst 16 active species. It wouldalso be of additional phytochemical interest since there are no previous reports of phototoxinsor photosensitizers from the mint family. In choosing a plant from a screening program for thepurification of the active component, the availability of the plant in quantity is again a factor.Samples of E. ciliata could be obtained in quantity as itis a commonly occurring shrub in Yunnan province andother parts of southern China (Steward, 1958).The Chinese name for Elsholtzia is 'xiang ru'( ). It was cited in Ben. Cao Gang Mu (Li,1979), the first Chinese materia medica, and theillustration is shown in Figure 3.1. In traditionalmedicine, the plant was used as a tonic against generalalterations to the body nature such as suffering from thechills (Huang, 1984). The common use of the plant intreating the cold and flu is probably related to this view.56The genus Elsholtzia has an Old World distribution with approximately 35 species of annuals,perennials and shrubs (Everett, 1981). Ten species and six synonyms were entered in theDictionary of Chinese Medicinal Herbs (New Jiangsu Hospital, 1977). These are listed inTable 3.1.In the orginal plant collection, a sample of Elsholtzia was collected as E. rugulosabecause it is cited for use in the treatment of the cold and flu. Leaves and twigs were collectedfrom a shrub that was not in flower. This sample proved to be the most active in the antiviralscreening. Later collections of larger quantities of the plant material for chemical purificationwere made by the Phytochemistry Laboratory of Kunming Institute of Botany. With the plantin flower, the identification was revised to E. ciliata. Both species are shrubs, with E. ciliatadescribed as being from 30-100 cm in height and E. rugulosa being 50-100 cm. The chemicalpurification process of the active compound was carried out with samples identified as E.ciliata. This species is also documented for usage in the treatment of cold and flu (JiangsuNew Hospital, 1979). Judging from the number of synonyms in the botanical literature onElsholtzia , it seems that the taxonomy within the genus is not well defined Considering thatin the medicinal literature survey, the name 'xiang-ru' applies to Elsholtzia in general, and theextracts of three species (E. blanda, E. ciliata, E. splendens ) showed similar UVA-enhancedantiviral activity, the question of the taxonomic distinction between E.ciliata and E. rugulosawas not considered to be critical in the search for an antiviral compound.57**E. blanda (Benth.) Benth.shrub, 100-150 cm'dien xiang ru' (dien = colloquial name for Yunnan)E. bodneri Vant.**E. ciliata (Thunb.) Hylandshrub, 30-100 cmE. cypriani (Pavlov.) Wu et Chow**E. densa Benth.annual, 10-50 cm'tu xiang ru' (tu = earth, soil)*E. flava (Benth.) Benth.shrub, 100-200 cm'da yeh xiang ru' (da yeh = big leaf)E. fructicosa (D. Don) Rehd.*E. penduliflora W.W. Smithshrub, 100-200 cm= E. cristata Willd.= E. patrini (Lepech) Garke= E. communis (Coll. et Hemsl.)Diels= E. polystachya Benth.*E. rugulosa Hemsl.shrub, 50-150 cm'xi zhou xiang ru' (xi zhou = fine wrinkle)**E. splendens Nakai ex Maek.^= E. haichowensis Sunperennial, 30-50 cm'xiang ru'^ = E. loeseneri Hand.-Mazz.Table 3.1Species of Elsholtzia Used in Chinese Medicine** assayed for and showed antiviral activity* potential assay candidate in Yunnan pharmacopoiea but not collected.58The chemical nature of the active component of E. ciliata was unknown and theapproach to the isolation was to use the process of bioassay-guided phytochemicalfractionation. There have been previous analyses of the chemistry of Elsholtzia species. Theliterature reporting the chemistry of Elsholtzia is listed in Appendix II. The majority of thestudies were done in India, China and Russia. In the NAPRALERT database (NAPRALERT,1992), over 90% of compounds reported from Elsholtzia are mono- and sesquiterpenes fromthe essential oil. Bisht et al. (1985) summarized the components of the essential oils of variousElsholtzia species and these include: terpenoids, unsaturated alcohols, carboxylic acids, andesters. Major components of the oils have been reported to be acylfuran derivatives such aselsholtzia ketone and dehydroelsholtzia ketone (Kobold et al., 1987). One report (Park et al.,1985) cited in NAPRALERT identified four flavonoid compounds from E. ciliata in Korea.Two other Korean reports (Chi and Lee, 1981; Han et al., 1981) were cited with conflictingresults regarding the absence of presence of saponins, and sterol and/or triterpene moieties inthe entire plant. Alkaloids are reported to be absent from E. patrini (syn. ciliata) (Woo et al,1978) and E. splendens (Chi and Lee, 1981). Since antiviral activity has been found in everymajor chemical group of compounds (Vanden Berghe et al., 1985; Roberts, 1988; Hudson,1990), the isolation of an active component has to take the empirical approach of identifying anunknown, by determining which fraction(s) contains the active component through bioassayafter each chemical separation. Hostettmann et al. (1991) generally summarized the process ofisolating bioactive constituents from medicinal plants in their review of the developments inpreparative separation techniques.The activity-guided fractionation of Elsholtzia ciliata extracts was divided into twoparts. The initial fractionation indicated the presence of more than one active component and asecond procedure was developed for isolating one of them.59Materials and MethodsAntiviral bioassaysAll fractions obtained from separation procedures were assayed for activity againstSindbis virus in the presence and absence of UVA light following the procedure described inthe Materials and Methods section of Chapter II. The virus / extract mixture was exposed to 30min of UVA prior to addition to the cell layer.Fractions were dried to residues first under reduced pressure at below 40°C then undera stream of nitrogen gas. The residues were weighed and redissolved in methanol to knownconcentrations for assay. With fractions that were not completely soluble in methanol,acetonitrile was used instead, or dimethyl sulfoxide (DMSO) was added. The fraction wasdiluted into PBS for assay such that the solvent concentration was never above 5% and theDMSO 0.1%. These concentrations have been shown to have no effect on the cells or virus.Crude extract preparationsAir-dried leaves and twigs of Elsholtzia ciliata (Thunb.) Hylander were obtained fromthe Kunming area of Yunnan. The first two samples were collected from the Forestry InstituteReserve at An-Ning 40 km. from Kunming. The latter samples were collected at HeilongtanPark in Kunming. The plant material was ground to a powder using a Wylie mill. The powderwas extracted exhaustively with methanol, with cold extraction followed by repeatedpercolations. Extracts were concentrated under reduced pressure at below 40°C. The crudeextract used in the second separation described (B) was obtained as a freshly extracted residuefrom the Phytochemistry Laboratory of the KIB. It was air-shipped from Hong Kong toVancouver.60Chemical SeparationsLiquid-liquid partition chromatographycrude methanolic extract : 5x volume H2Opartition successively in order of increasing polarity with:hexanesdiethyl etherchloroformethyl acetatebutanolFlavonoid extractionEthanolic crude residue was washed with boiling H2O, filtered and separated with H2Osaturated butanol. The butanol fraction was dried and taken up with methanol for assay.Column liquid chromatography (CC) and thin layer chromatography (TLC)Separation of active solvent fractions were tried with chromatographic systems usingthe following stationary phases, and different organic solvent gradients :silica^column (Silica gel 60, 70-230 and 230-400 mesh)TLC (Merck Kieselgel 60)alumina^TLC (Eastman Kodak 6062)polyamide TLC (MN-Polyamid DC 6.6)Sephadex LH2O^column (Pharmacia)acetylated polyamide^column (MN-Polyamid SC6-AC)61Charcoal separationAn active column fraction was swirled with activated charcoal powder and washedrepeatedly over celite through a sintered glass funnel. The eluted fraction was concentrated forassay.High performance liquid chromatography (HPLC)Active column fractions were further separated on HPLC using a Waters 660Econtroller, and the Waters 994 photodiode array detector (Millipore). The UV detector was setto scan for absorption at 330nm, as UVA-enhanced active column fractions have shownabsorption at this wavelength. Normal phase separations were done using an analytical MerckHibar Lichrosorb Si60 column (5 pt,M particle size), with chloroform as the major eluatingsolvent. Methanol was added to increase polarity for both isocratic and gradient systems in theprocess of optimizing separation. Up to 0.2% triethylamine or up to 1% acetic acid were addedas weak ionic buffers. Reverse phase separations were done using an analytical VarianMCH10 column (10 tiM particle size), with acetonitrile (MeCN): water isocratic or gradientelution systems. In buffered systems, 1% acetic acid was added. Flow rate was 1 ml/min.Structural Analyses Ultraviolet SpectroscopyUV spectra of liquid column fractions were obtained on the Pye Unicam SP8-100 UVspectrophotometer. Spectra from HPLC fractions were obtained during the separations on theWaters 994 photodiode array detector.62Infrared Spectroscopy (IR)IR spectra were obtained by C. De Soucy-Brau on the Perkin-Elmer 1710 infraredspectrophotometer with Fourier transform, using chloroform as solvent, in the ChemistryDepartment, UBC.Mass spectroscopy (MS)Electron impact mass spectra were obtained using both gas chromatography (GC)electron impact MS, and from solid probe samples on a Finnigan 1020 automated GC/MS.High resolution mass spectroscopyHigh resolution MS was done by the Mass Spectrometry Centre at the ChemistryDepartment of UBC and the fragmentation analyzed with the DS-55 MS data system.Proton nuclear magnetic resonance spectroscopy ( 1 H-NMR)Proton NMR spectra were obtained at 400 MHz, using CDC13 or CD2C12 as solventand tretramethylsilane (TMS) as internal standard, on the Brucker instrument at the NMRfacilities of the Chemistry Department of UBC.63ResultsChemical separation and purificationLiquid-liquid solvent partitioningIn solvent partitioning of the crude extract, there was activity in the three least polarsolvent fractions. The results are shown in Table 3.2Table 3.2Concentrations of Solvent Partition Fractions Active Against Sindbis Virus with UVA LightFraction Active Concentration µg/mlmethanolic crude 0.3hexane < 0.15ether < 0.15chloroform 0.65ethyl acetate not activebutanol not activeFlavonoid extraction and charcoal separation64The fractions obtained from these procedures were not active.Fractionation proceduresA. Starting with crude extraction of 400 g of dry plant material, followed by solventpartitioning.active ether solvent residueacetonitrileinsoluble solubledryether65soluble insoluble5% NaHCO3bicarbonate^etherneutralize to pH 7.5choloroformH2O^chloroformflash silica column95:5 chloroform / methanol+ 0.3% triethylamineHPLCnormal phase columnMerck Hibar Lichrosorb Si60chloroform + 0.1% acetic acidFractionation procedure A was developed from empirical trials for better separation ofactive components A single active component was not isolated from the procedure. Twoactive HPLC fractions were obtained with normal phase buffered conditions. The retentiontimes (RT) of these two fractions differed by 18 minutes, indicating a difference in polarity.The two active peaks representing these fractions are indicated in the HPLC chromatograph inFigure 3.2. It was seen on TLC that these fractions had multiple components, but furtherseparation was stopped at this point as the yield of this active material was less than 1 mg fromthe 400 g dry plant sample. Any further separations would result in insufficient quantities forfurther structural analysis.B. Starting from methanolic crude extract residue from 8 kg of fresh plant materialobtained from the Phytochemistry Laboratory of KIB.1:1 methanolic crude extract / H2Ohexane partitionsilica column95:5 hexane / etherorange color fractionsilica columnhexaneUV fluorescent fractionacetonitrile(active fraction)HPLCreverse phase columnVarian MCH107:3 acetonitrile / H2Opetroleum ether660^10^ 20^ 30^ 40M 18134 151^2 34567^91 0 1 11 2 17^ 18^191 "^' 1^ ' 1 '^f "^ 10^10 20^ 30 40Retention Time (Min)Figure 3.2: Normal Phase HPLC Chromatograph of Elsholtzia Fraction(Peaks representing fractions showing antiviral activity indicated by arrows)67Fractionation procedure B was developed using a much larger quantity of material forseparation. It resulted in the isolation of an active compound from the most nonpolar fractionof the extract. The active components that exhibited a more polar nature have not beenpurified.A single peak indicated by the HPLC chromatograph was collected and found to beactive ( RP MCH10 column, 7:3 CH3CN : H2O, 1 ml/min flow rate, RT 13 min, UV 350 nmdetection). The purification of the peak by repeated HPLC injection is shown in thechromatographs in Figure 3.3. The yield was 700 pg from 8 kg of fresh plant material, or 87parts per billion (ppb).6820 30 4010 12C.2(110^min^20Retention TimeRetention Time (Min)B.1816^ 20^22^24^2410.00 --- 22.00 min16 22212423^253456201011 1231415 6" 27^2°^29^3030211A.Ml2 3Figure 3.3: Separation of Active Compound By Reverse Phase HPLCA. UV absorption peak of compound targetted for isolation in HPLCchromatograph (indicated by arrow).B. UV spectra of individual compounds from series of chromatographic peaks.C. Chromatograph of isolated compound (UV spectrum shown in Figure 3.5).69Structural identificationThe active compound was identified as the polycyclic aromatic hydrocarbonfluoranthene, Ci6H10. The chemical structure is shown in Figure 3.4.Figure 3.4: Chemical Structure of FluorantheneUltraviolet spectroscopyThe UV spectrum was obtained from the HPLC photodiode array detector and shownin Figure 3.5. Molecular extinction coefficients (E) were calculated for the absorption maxima(X max) from absorption units using Beer's Law and compared with published values forfluoranthene in Table 3.3.70Figure 3.5: UV Spectrum of Purified FluorantheneTable 3.3Extinction Coefficients from Ultraviolet Spectra of Fluoranthenesam le values^unshed values *n mX m ax A UcalculatedE x maxnme236 .510 50000 235 39800277 .224 21960 276 22400287 .328  32160 287 31600323 .060 5880 323 5750343 .080 7840 342 7760359 .080 7840 358 7950AU = absorption unit from Waters 994 photodiode array detector,(concentration of sample injected = 1 mg/ml)* from Morton (1975) Biochemical spectroscopy.= extinction coefficient^X max = absorption maximum71IR SpectroscopyThe IR spectrum indicated the presence of aromatic components in the region of 700-800 cm -1 and 1200 cm -1 The spectrum is shown in appendix III.Mass spectroscopyThe EI spectrum of a solid probe sample with the relative abundance of fragments isshown in Figure 3.6.High resolution mass spectroscopyThe molecular ion mass of 202.0786 was matched to the mass of 16 carbon and 10hydrogen atoms, with 0.4% deviation. The fragment analysis is shown in Appendix IV.Proton nuclear magnetic resonance spectroscopyThe 1 H-NMR spectra of the purified sample and the authentic sample from AldrichChemicals of fluoranthene are shown in Figure 3.7.7220260 140^160^180^200M/E^40 80^100^120101888475150111^ 174II1^I 1231^1111 1111 157^I^dill.^187^IIerilt , 1 0,, ,-1 1 1^I MI I^et't^„ r i „ r^el55693101III4950.0 —100.0—41Figure 3.6: EI Mass Spectrum of Purified Fluoranthene(Solid probe sample on Finnigan 1020 automated GC/MS)E/I MS m/z (relative abundance): 204 (1.47), 203 (17.84), 202 [M+ , C161-110] (100),201 (14.45), 200 (25.46),174 (5.91), 150 (5.98), 111 (5.22), 101 [C81-15] (72.28), 100(60.28), 99 (20.05), 88 [C7H4] (57.74), 87 (32.31), 75 (23.25), 55 (28.76), 49 (62.49),41 (31.11).The fragment assignments were obtained from the high resolution MS fragmentanalysis shown in Appendix IV.7374Figure 3.7: NMR Spectra of Purified (A) and Authentic (B) Samples of Fluoranthene.Spectral assignments are shown in the following table:Table 3.41H-NMR Spectral Assignments of FluorantheneProton Chemical Shift (ppm) Coupling Coupling Constants (Hz)8 1 = 8 6 7.97 d J = 7.008 2 = 8 5 7.66 dd J = 7.00, 8.138 3 = 8 4 7.87 d J = 8.1387=810 7.95 dd J = 3.12, 5.5188=89 7.40 dd J = 3.12, 5.51Carbon positions are shown^d = doublet dd = doublet of doubletin Figure 3.4.Coupling constants were calculatedfrom a 10Hz/cm expansion.75Virus UVA DarkSINV 0.01 5.0MCMV 1.0 10.0olio N.A. N. A .Antiviral activity of fluorantheneThe antiviral activities of fluoranthene with and without exposure to UVA are shown inTable 3.5. Both purified and authenic samples were tested and showed the same potency.Table 3.5Minimum Active Antiviral Concentrations of Fluoranthene76DiscussionThe bioactivity-guided empirical approach was used to separate the unknown activecomponents. The basic scheme was to separate the plant extract by chemical nature and pursuefurther separation of the most active fraction after each step. Preliminary separation by natureof polarity was done with successive partitioning of crude extract against solvents of increasingpolarity. The antiviral activity was found to be located in the three least polar solvent fractions.Column chromatography was continued with one fraction and in a number of trials, the activitywas found to spread over a series of fractions with several different chromatographic systemsand conditions. Procedure A was developed first in the chemical separation of the activefraction in Elsholtzia ciliata . Silica is the most commonly used chromatographic stationaryphase material. It is applicable in the separation of a wide range of compounds but itsadsorptive nature will tend to retain some chemical types and give poor separation.Compounds of ionic nature or those with polar functional groups are known to separate poorlyand give tailing bands in liquid-solid chromatography (Snyder and Kirkland, 1979). In aneffort to achieve better resolution of the activity than that obtained using silica systems, otherstationary phase sorbent materials were selected to cover a range of characteristics that areconsidered suitable for the separation of different types of chemical compounds (Touchstoneand Dobbins, 1983). Alumina, like silica, separates by order of polarity but can give betterseparation of some compounds such as bases and quinolines. Polyamide and acetylatedpolyamide are considered to have less adsorptive properties and may give better resolution ofcompounds that have a tendency to be retained. Sephadex LH2O is a chemically modifieddextran polysaccharide used in gel filtration chromatography which separates more bymolecular size than by adsorption. Other chromatographic conditions such as columndimensions and solvent elution gradients were also varied. Significantly better separation ofthe active fraction was not obtained by modifying the adsorbent natures and solvent polarstrengths.77The approach of testing fractions obtained from following established separationprocedures for a group of potential bioactive compounds was tried. The flavonoid fractionwas not found to be active. The active fractions from solvent partitioning and preliminarycolumn separations were strongly pigmented by chlorophylls. Treatment with charcoal wasused for the removal of pigments, but assay of the remaining fraction showed that the activecomponent was retained by the charcoal. Therefore charcoal treatment could not be used as aseparation step.It was considered that the lack of resolution in chromatography was due to the chemicalnature of the active components. Since ionic compounds are known to give poor liquid-solidchromatographic resolution, the extract was separated into acidic, basic and neutral portionsfollowing classic organic chemical separation methods (Shriner et al., 1980). The activity wasfound to be in the acidic portion. Chromatography of the acidic portion showed betterresolution with the addition of a weak base or weak acid as a buffer in the solvent system.This supported the hypothesis that ionic components contributed to the poor chromatographicresolution. HPLC separations indicated that there was more than one active component. As itstands, the active constituents in the acidic fraction following procedure A have not beenisolated. It has been shown that ionic compounds inhibit Sindbis virus infection by affectingits binding to cell membrane receptors (Mastromarino et al., 1991). This inhibition would takeplace in the adsorption process. The Elsholtzia fractions were active in treatments prior tocellular entry of the virus that would include the adsorption process. Since the active fractionexhibited ionic properties, it is possible that the active component exhibits this type of action.When it was determined that the plant sample used to develop procedure A would beinsufficient to yield the active compounds in enough quantity for structural identification, alarger quantity of crude extract was obtained from Yunnan. With a larger quantity of extract, anonpolar active fraction in a workable quantity could be separated from the hexane portion of78the initial solvent partition. Procedure B was developed for the isolation of an active compoundand it did not require the pH partitioning and buffering treatments of procedure A. It is likelythat the more polar active component that has yet to be purified was responsible for the ionicbehavior of the active fraction in procedure A. The development of procedure A was the resultof addressing the chemical nature of this more polar component because it was present in ahigher concentration in comparison to the less polar active component. The presence of a verynonpolar active fraction had been observed in some of the chromatographic trials but itsconcentration in the smaller sample really did not allow for further purification. As it turnedout, the nonpolar active compound identified represented only 87 ppb of plant material.The purified active compound was identified as the polyaromatic hydrocarbon (PAH)fluoranthene, with the structural formula of C16H10• The fragmentation pattern of the massspectrum gave a 98% match with that of fluoranthene in the computerized National Bureau ofStandards Mass Spectra Library. High resolution MS was done to confirm the atomiccomposition. Since PAHs are not known plant compounds, other possible molecularcompositions containing nitrogen and/or oxygen were considered. The atomic formula offluoranthene gave the closest fit to the molecular ion mass of 202.0786. The other majorfragments also showed the closest fit with a compound composed of carbon and hydrogen.The low field shifts in the proton NMR spectrum indicated the presence of aromaticcomponents. The behavior in chromatography and the characteristics of the UV spectrumsuggested the possibility of photoactive plant compounds such as thiophenes, which are sulfurheterocycles. The UV spectra of acetylenic compounds often exhibit a series of peaks in the300-400 nm range with a maximum between 220-280 nm (Bohlmann et al., 1973). Thecharacteristic series of peaks in the acetylenes is attributed to the series of conjugated bonds.The spectra of PAHs, however, exhibit a very similar pattern. Aromatic rings have series ofconjugated bonds that have the same UV absorption characteristics (Morton, 1975). The79max and extinction coefficients calculated from absorption units of the spectrum from a purifiedsample matched published values for fluoranthene. Since fluoranthene has no substitutionfunctions, the IR spectrum only indicated the presence of aromatic groups.The chemical shifts and coupling constants obtained for the purified sample could notbe matched to any published NMR spectra for thiophenes or other aromatic structures. Whenother data indicated the structure of fluoranthene, the spectrum was compared to those offluoranthene in The Aldrich Library of NMR Spectra (Ponchart and Campbell, 1973) and TheSadtler Handbook of PNMR (Simmons, 1978). The chemical shifts were in the same fieldregion between S 7.15-8.0 ppm, the multiplets were more confused in the published spectra.The splitting patterns were also not the same as those analyzed by Heffernan et al. (1967).However, their spectrum was obtained at 100 MHz whereas the purified sample spectrum wasobtained at 400 MHz. A sample of fluoranthene was purchased from Aldrich Chemicals (F80-7) and subjected to the same 400 MHz NMR scan. A spectrum with the same shifts andsplitting patterns between S 7 and 8 ppm as the purified sample spectrum was obtained (Figure3.7). Fluoranthene is a bilaterally symmetrical molecule, therefore the spectral assignmentsshowed only five types in the ten protons.With the match of the NMR, Mass, and UV spectra and HPLC chromatograms of anauthentic fluoranthene sample to those of the purified compound, no further analyses weredone. Antiviral components from the more polar active fraction of the Elsholtzia extract haveyet to be isolated and identified. Their identification is also an interesting question in regard towhether photo-active plant compounds occur in Elsholtzia and the Lamiaceae.Fluoranthene is not known as a plant constituent but it was isolated from a plant extractusing bioassay-guided fractionation. It was found as a light-mediated antiviral activity targetcompound because it showed potent UVA-enhanced activity against Sindbis virus (10 ng/ml80with UVA and 5 µg/ml in dark). There were several other HPLC peaks within a 20 minuteretention range of fluoranthene (see Figure 3.3). They were not purified for identification butthose HPLC fractions also showed antiviral activity. The PAHs phenanthrene and pyrene(obtained from Dr. J. Kagan, Department of Chemistry, University of Illinois) were injectedon HPLC for comparison. They could be matched by retention time and by UV spectra topeaks in the active HPLC fractions. It is probable that a group of PAHs are present in the plantextract, and they could have also contributed to the light-mediated antiviral activity. Theantiviral mechanism of fluoranthene will be discussed in the next chapter, but how thiscompound came to be isolated from a plant sample has to be considered.It is possible that the source of the PAHs was from contamination in the purificationprocedure. Large volumes of solvents were used to process a large sample of plant extract anda compound present in less than 1 part per million (ppm) was isolated. With separation andanalytical techniques capable of purifying trace components, it has become more of apossibility that impurities from materials used in the processing can be accumulated and befound in an identifiable quantity. The major solvent used in this procedure consisted of amixture of n-hexanes. The impurities remaining after the evaporation of 4 liters of hexanesunder reduced pressure was submitted for NMR (De Soucy-Brau, Chemistry DepartmentUBC, unpublished data). While there was a peak in the aromatic region of the spectrum, it didnot match any of those obtained for fluoranthene, nor was the spectrum complex enough toindicate the possible presence of PAHs. As another check, a small amount of the crude extract(60mg) was processed using only 60 ml of hexanes. The fluoranthene peak was obtained onthe HPLC trace from this treatment, further indicating that it was not from the accumulation ofimpurities from hexanes. Another piece of evidence supporting this conclusion is that UVfluorescent nonpolar active fractions had been detected in earlier chromatographic trials usingother solvent fractions of the plant sample. Even though they were not chemically identified, itis likely that they consisted of the PAHs and were not solvent-related.81Polyaromatic hydrocarbons have been found in foliage as well as in crops (Buckley,1987). PAHs are generated by the incomplete combustion of fossil fuels, especially coal(Williamson, 1973), and are found throughout the environment in air, water, sediment andsoil. The levels in the environment can be correlated to levels of human activities (Jones et al.,1989). Analyses of plant foliages have been used to monitor levels of anthropogenic chemicalspresent in the environment (Eriksson et al., 1989). Wild and Jones (1991) extracted total PAHcontents in the range of 5 - 16 [tg/kg fresh weight from carrots. Jones et al. (1992) archivedannual vegetation samples from 1965 to 1989 for analysis of levels of polychlorinatedbiphenyls and PAHs. They found total level of 13 PAHs to range from 187 to 472 ng/kg dryweight. The level of fluoranthene ranged from 9 to 31 ng/kg dry weight. In comparison, theyield from Elsholtzia ciliata of 87 lig/kg of fresh plant material was higher but of a similarmagnitude to PAH levels reported from plant sources.If the explanation for the occurrence of PAHs in the Elsholtzia samples involvesaccumulation of environmental pollutants, then such compounds might be expected to be foundubiquitously in the other species tested. In a study of chlorinated hydrocarbons, Calamari et al.(1991) found them to be present in foliage in all parts of the world. Furthermore, there is alinear relationship between concentrations of chemicals in foliage and in air. The question thenarises as to why the antiviral activity shown by the PAHs in Elsholtzia ciliata was not seen inall the Yunnan plants screened, or at least from samples collected in the same area. The level ofPAHs present generally in plants may not be high enough to generate antiviral activity. In E.ciliata , besides the PAHs, there was another active component that has not been identified.The high activity of the plant extract in the screening program was probably from the combinedeffect of several compounds, and the first compound to be identified from bioassay-guidedfractionation was a PAH. Another mint plant (Stachys ) also showed UVA-enhanced activityin the antiviral screening; the active component(s) had not been identified. It would be82interesting to see if there is also a combination of PAHs and other active components present inStachys kouyangensis.It is also possible that Elsholtzia accumulates a higher concentration of PAHs.Buckley (1982) had found variation between species when examining the accumulation ofPCBs in foliage. In a review, SchOnherr and Riederer (1989) discussed the properties ofchemicals and plant cuticles affecting the foliar uptake of chemicals. They considered the leafsurface to be a major interceptor of airborne pollutants by calculation of geometric area alone.Riederer (1990) followed with a theoretical model of the foliage / atmosphere system tocalculate the partition coefficients for reference chemicals with different components in the air-to-vegetation transfer of persistent organic chemicals. The values show that the lipophilicportion of the foliage, representing the cuticle in the model, can effectively scavenge lipophilicmolecules from the surrounding atmosphere. It is thus possible that foliage with morelipophilic components will also have a greater tendency to accumulate airborne lipophilicmolecules like the PAHs.Plants of the mint family often have high contents of lipophilic molecules in trichomes,as demonstrated by the fact that they are the sources of many aromatic oils (Baumgardt, 1982).This family characteristic may lead to a higher accumulation of PAHs, and other pollutantmolecules in Elsholtzia. Four species were assayed and three of them showed UVA-enhancedantiviral activity. The species E. densa showed more moderate activity which was not UV-mediated. This is an annual plant whereas the other three are perennial shrubs. It is likely thatthis difference arises from greater sequestering of environmental compounds by perennialplants. Since many PAHs have been shown to be carcinogenic (Jones, 1982), their possibleaccumulation in the Lamiaceae might raise health-related concerns regarding the popularconsumption of herbs from the mint family. The topic of differential accumulation needsfurther investigation.83It was an unexpected outcome that the compound isolated from this bioassay-guidedsearch for active molecules in a medicinal plant species is probably not synthesized by theplant. This occurred because the photo-activity of fluoranthene met the selection criteria of theUVA-mediated antiviral screening program. The pharmacognosist should be aware of that theprocess of isolating a bioactive natural products can lead to the identification of anthropogeniccompounds. The presence of anthropogenic contaminants in the environment and in organismshas been of concern as possible threats to human health because of their known or potentialtoxicity and carcinogenicity. Since many of these chemicals display bioactivity, it is perhapsnot surprising to find a "pollutant" in a search that targets by detection of bioactivity. As manyplants provide food and medicines, the distinction between environmental contaminents andphytochemicals in plants has significant implications in human health. Plants are also animportant component of the environment as a whole, and further investigations into therelationship between environmental chemicals and phytochemistry may contribute to ourunderstanding of human impact on the global environmental quality.84Chapter III. ReferencesBaumgardt, J.P. (1982) How to Identify Flowering Plant Families. Timber Press, Beaverton,pp. 162-163.Bisht, J.C., Pant, A.K., Mathela, C.S., Kobold. U. and Vostrowsky, O. (1985) Constituentsof essential oil of Elsholtzia strobilifera. Planta Medica 51, 412-414.Bohlmann, F.T., Burkhard, T. and Zdero, C. (1973) Naturally Occurring Acetylenes.Academic Press, New York, 547 pp.Buckely, E.H. (1982) Accumulation of airborne PCBs in foliage. Science, 216, 520-522.Buckley, E.H. (1987) PCBs in the atmosphere and their accumulation in foliage and crops.In: J.A. Saunders, L. Kosak-Channing and E. Conn (eds.) Phytochemical Effects ofEnvironmental Compounds. Recent Adv. in Phytochem. Vol. 21, Plenum Press, NewYork, pp. 175-201.Calamari, D., Bacci, E., Focardi, S., Gaggi, C., Morosini, M., and Vighi, M. (1991) Role ofplant biomass in the global environmental partitioning of chlorinated hydrocarbons.Environ. Sci. Technol., 25(8), 1489-1495.Chi, H.J. and Lee, S.Y. (1981) Phytochemcial screening of Korean Medicinal Plants(III).[cited in NAPRALERT], Ann Rep. Nat. Prod. Res. Inst. Seoul Natl. Univ. 20,38-41.Eriksson, G., Jensen, S., Kylin, H. and Strachan, W. (1989) The pine needle as a monitor ofatmospheric pollution. Nature, 341, 42-44.Han, B.H., Lee, E.B. and Woo,W.S. (1981) Screening of saponins in the plants. [cited inNAPRALERT], Ann. Rep. Nat. Prod. Res. Inst. Seoul Natl. Univ. 20, 49-54.Heffernan, M.L., Jones, A.J. and Black, P.J. (1967) Proton magnetic resonance studies ofnon-alternant hydrocarbons II. fluoranthene and benzo(ghi)flouranthene. Aust. J.Chem. 20, 589-593.Hostettmann, K., Hamburger, M., Hostettmann, M. and Marston, A. (1991) Newdevelopments in the separation of natural products. In: N.H. Fischer, M.B. Isman andH.A. Stafford (eds.) Modern Phytochemical Methods. (Recent Advances inPhytochemistry, Vol. 25), pp. 1-32.Huang, S.Y. (1984) Introduction to Chinese Medicine. [in Chinese], Ba De EducationalPublications, Taipei, 150 pp.Hudson, J.B. (1990) Antiviral Compounds from Plants. CRC Press, Boca Raton, 200 pp.Jones, K.C., Stratford, J.A., Waterhouse, K.S., Furlong, E.T., Giger, W., Hites, R.A.,Schaffner, C. and Johnston, A.E. (1989) Increases in polynuclear aromatichydrocarbons of an agricultural soil over the last century. Envir. Sci. Technol. 23, 95-101.85Jones, K.C., Sanders, G., Wild, S.R. Burnett, V. and Johnston, A.E. (1992) Evidence for adecline of PCBs and PAHs in rural vegetation and air in the United Kingdom. Nature356, 137-139.Jones, P.W. (1982) Polynuclear aromatic hydrocarbons. In: M.C. Bowman (ed.) Handbookof Carcinogens and Hazardous Substances. Marcel Dekker, New York, pp. 573-639.Kobold, U., Vostrowsky, 0., Bestmann, H.J., Bisht, J.C., Pant, A.K., Melkani, A.B. andMathela, C.S. (1987) Terpenoids from Elsholtzia species; H. constituents of essentialoil from a new chemotype of Elsholtzia cristata. Planta Medica 53, 268-271.Li, S. (1979) Ben Cao Gang Mu. Compiled in 1578, [in Chinese], People's HealthPublications, Chengdu, 1432 pp.New Jiangsu Hospital (1077)Dictionary of Chinese Medicine. Vol. 1 and 2 (1977) [inChinese], Shanghai Science and Technology Publications, Shanghai, 2754 pp. + 764pp. appendix.NAPRALERT database (1992) issue 2, maintained by the Program for Collaborative Researchin the Pharmaceutical Sciences, Dept. of Medicinal Chemistry and Pharmacognosy,College of Pharmacy, University of Illinois at Chicago.Mastromarino, P., Conti, C., Petruziello, R., Lapadula, R. and Orsi, N. (1991) Effect ofpolyions on the early events of Sindbis virus infection of Vero cells. Arch. Virol. 121,19-27.Morton, R.A. (1975) Biochemical Spectroscopy. Vol. I.,Wiley, New York, 381 pp.Park, J.H., Woo, W.S. and Shin, K.H. (1985) Flavonoids of Elsholtzia ciliata [cited inNAPRALERT], Korean J. Pharmacog. 16 (1), 43-A.Ponchart, P.J. and Campbell, J.R. (eds.) (1974) The Aldrich Library of NMR Spectra. Vol.IV. Aldrich Chemicals, Milwaukee, pp. 35.Riederer, M. (1990) Estimating partitioning and transport of organic chemicals in thefoliage/atmosphere system: discussion of a fugacity-based model. Environ. Sci.Technol. 24, 829-837.Roberts, S.M. (1988) Design of antiviral agents other than nucleoside analogues. In: E. DeClercq and R.T. Walker (eds.) Antiviral Drug Development: A MultidisciplinaryApproach. NATO ASI Series A: Vol. 143, Plenum Press, New York, pp. 37-54.Schônherr, J. and Riederer, M. (1989) Foliar penetration and accumulation of organicchemicals in plant cuticles. Rev. Env. Contam. Toxicol., 108, 1-70.Shriner, R.L., Fuson, R.C., Curtin, D.Y. and Morrill, T.C. (1980) The SystematicIdentification of Organic Compounds. 6th Edit., Wiley, New York, 604 pp.Simmons, W.W. (ed.) (1978) The Sadtler Handbook of PNMR. Sadtler Reserach,Philadelphia, pp. 101.Snyder, L.R. and Kirkland, J.J. (1979) Introduction to Modern Liquid Chromatography, 2ndEdition. Wiley, New York, pp.353.86Steward, A.N. (1958) Manual of Vascular Plants of Lower Yangtze Valley,China.Oregon State College, Corvalis, 612 pp.Touchstone, J.C. and Dobbins, M.F, (1983) Practice of Thin Layer Chromatography, 2ndEdition, Wiley, New York, 405 pp.Vanden Berghe, D.A., Vlietinck, A.J. and Van Hoof, L. (1985) Present status and prospectsof plant products as antiviral agents. In: A.J. Vlietinck and R.A. Dommisse (eds.)Advances in Medicinal Plant Research. Wissenschaftliche, Stuttgart, pp. 47-99.Wild, S.R. and Jones, K.C. (1991) Studies on the polynuclear aromatic hydrocarbon contentof carrots (Daucus carota ). Chemosphere 23(2), 243-251.Williamson, S.J. (1973) Fundamentals of Air Pollution. Addison-Wesley, Reading, 472 pp.Woo, L.K., Yun, H.S., Chi, H.J., and Woo, W.S. (1978) Occurrence of alkaloids in Koreanmedicinal plants. [cited in NAPRALERT] Soul Taehakkyo Saenyak Yonguso Opjukjip17, 17-19.87Chapter IV.Structure - Activity Relationship Studies of the Antiviral Compound HypercinIntroductionThe aim of this project was to conduct a thorough study of a biologically activecompound, beginning from a screening search and continuing with the isolation of the activecompound to the investigation of its nature of action. Medicinal plants from Yunnan, China,were screened for antiviral activity and the species Elsholtzia ciliata was selected for theisolation of an active component. Ideally, the nature of action studies would have beencarried out with the purified compound. However, during the empirical development of thepurification process, it appeared that the time it would take to follow the original plan wouldbe of concern. It was decided to conduct the nature of action studies with an alternativeknown antiviral plant compound while the purification of the Elsholtzia component was stillin progress. Hypericin is a photosensitizer antiviral which has been isolated from Hypericum(Hypericaceae). Since the activity of Elsholtzia was shown to be light-mediated, there is asimilarity in the photosensitization nature of the activity. Hypericum japonicum Thunb. andH. patulum Thunb. are both used in Yunnan to treat hepatitis and were on the list compiledof candidates for antiviral screening (Table 1.1, Chapter I) even though plants were notcollected from Yunnan in this survey. In view of the photo-active nature of the compounds,it is useful to begin with a general discussion of photosensitizer compounds.88Photosensitzer CompoundsA photosensitizer is a chemical that can have an electron promoted to a higher energyorbital with the absorption of a photon. The excited state molecule in turn reacts with asecond molecule. Reactions where electronically excited molecules generate radicals whichact as intermediates are classified as type I, and reactions where only electronically excitedmolecules act as intermediates are classified as type II (Gollnick, 1968). Frequently inbiological systems the intermediate reacts with oxygen and a reactive oxygen species isgenerated. Singlet oxygens are reactive oxygens with promoted electron energy states, andthey can be generated by an input of energy (Halliwell and Gutteridge, 1989). Other reactiveoxygens are generated via free radicals, the addition of a single electron generates thesuperoxide anion, and the addition of one more electron produces the peroxide ion. Thesetwo types of reactions do not necessarily occur exclusive of each other. Plantphotosensitizers have commonly exhibited type II reactions with the formation of singletoxygen (Hudson and Towers, 1991). In the cascading process, when the reactive moleculesaffect a target molecule and produce a biological effect, the result is considered aphotodynamic reaction (Towers, 1984; Spikes, 1989). The activity can be mediated by longwavelength ultraviolet UVA (320-400 nm) or visible light. Some common photosensitizersare pigments, ketones, quinones and aromatic molecules (Foote, 1987). The requirement oflight for the activity or enhancement of activity for these compounds can be used as a point ofexperimental manipulation in studying their modes of action.Free radical-mediated reactions are part of normal cellular metabolic functions butthese type of reactions can also cause harmful biological effects in the form of membrane orprotein damage, or lipid peroxidation (Fantone and Ward, 1985). The biological activity ofthe photo-active compounds has potential therapeutic application when these types of tissuedamage are selectively targeted at micro-organisms, pests or tumor cells. Viruses in89particular are also susceptible to effects of reactive molecules on the nucleic acid strands.Photosensitizers have been shown to have antimicrobial, antiviral and insecticidal effects(Marchant, 1987). Many different classes of chemicals have been found as naturallyoccurring phototoxins. The major classes are: the polyketides (polyynes, thiophenes,quinones and chromenes), cinnamate derivatives (coumarins and furanyl compounds), andalkaloids (derivatives of tryptamine, phenylalanine, tyrosine and anthranilic acid). Theactions and therapeutic prospects of plant photosensitizers were reviewed by Hudson andTowers (1991). The antiviral properties of three groups of compounds were reviewed byHudson (1989). The activities of thiophenes and polyynes are attributed to singlet oxygendamage to viral membranes (Hudson and Towers, 1988). The generation of singlet oxygen inUVA by thiophenes and related compounds has been demonstrated (Garcia et al, 1984).Membrane damage by the acetylenic compounds a-terthienyl and phenylheptatriyne has alsobeen shown (MacRae et al., 1980). Furanyl compounds such as furanocoumarins, appear toact through the formation of covalent adducts with viral nucleic acids (Hradecna and Kittler,1982; Hudson et al., 1988). The compounds are thought to intercalate with the nucleic acidsat the pyrimidine bases. The resulting adducts would prevent the execution of normal genetictranscription or translation (Song and Tapley, 1979; Cimino et al., 1985). The action of thefuranoquinoline alkaloids is similar to that of the furanocoumarins (Pfyffer et al., 1982). The13-carboline alkaloids, however, do not cause cross-linking with viral DNA (Altamirano etal., 1986). They probably do act on the nucleic acids as they have been shown to haveinteractions with DNA in studies that did not consider the role of light (Duportail and Lami,1975; Hayashi, et al., 1977), and to cause UVA-mediated chromosomal damage in cells(Towers and Abramowski, 1983).In considering the therapeutic applicability of photosensitizers, the question arises asto how the light is administered to activate the compounds. The situation is simple in thecase of topical applications. Psoralen-UVA therapy has been used in the treatment of the90skin affliction psoriasis (Parrish et al., 1974; Gupta and Anderson, 1987). Aphotochemotherapy system was also developed for the treatment of cutaneous T-celllymphoma with methoxypsoralen with extracorporeal photo-activation of patients' bloodfractions (Edelson et al., 1987). It is now also possible to direct light to specific internaltissues with the use of laser fiber-optic technology. Coupled with the ingestion of aphotosensitive chemical, the requirement of light for activation then becomes a tool forselectively targeting tumor tissues for sites of action (Douglas et al., 1981). A number ofstudies have also looked at the use of photosensitizers in the sterilization of blood products,especially with regard to the inactivation of viruses which may not be removed by filtrationmethods applicable to other micro-organisms (Matthews et al., 1988; Nyendorff et al., 1990).The sterilization may be achieved with a lower concentration of a photosensitive than a non-photosensitive compound because the potency is enhanced by light. The presence of acompound that is also inactive in the dark in low concentration may be considered clinicallymore suitable for blood products used in transfusion. Controlling the time or location ofactivity by manipulating the application of light will likely lead to the development of moreinnovative therapeutic applications with photosensitizers.The antiviral compound fluoranthene, isolated from Elsholtzia, has been shownpreviously to have photo-activity against a number of aquatic organisms such as fish,mosquito larvae and brine shrimp (Kagan, 1985), indicating a general toxicity to eukaryotecells. It causes hemolysis of human erythrocytes with UVA exposure through action on thecell membrane (Wang and Kagan, 1989). It also inactivates Escherichia coli in the presenceof UVA. Tuveson et al. (1987) tested its effect on DNA repair deficient mutant E. colistrains and on the transforming activity of Haemophilis influenzae transforming DNA toshow that DNA can be a target of action. Induced mutation was not shown with a histidinelocus reversion assay, but this does not necessarily rule out the possibility of mutagenicactivity. In the same study, photo-oxidation experiments showed the generation in light of91singlet oxygen by fluoranthene in both organic and aqueous media. There is also evidencefor the formation of superoxide anions, which is known to lead in turn to the generation ofreactive hydrogen peroxides. The exact mechanism of the photodynamic action offluoranthene is thus complex, and it is possible that both singlet oxygen and superoxideproduction take place. Because of its phototoxicity and possible mutagenicity, the antiviralactivity of fluoranthene may not have therapeutic potential. While the activity shown wassignificant, it was likely to have arisen from a non-virus specific oxygen-mediated attack onmembranes. Even though fluoranthene is regarded as one of the more benign PAHs in regardto carcinogenicity, its role as an environmental pollutant is still to be estimated because of itsphototoxicity and undetermined mutagenicity.Investigations of the mechanism of action of a photosensitive antiviral plantcompound was continued with the quinonic compound hypericin. The studies concerningstructure-activity relationships is covered in this chapter. The studies concerning the site andmolecular target of action is covered in the following chapter.HypericinSpecies of the genus Hypericum have been documented as traditional medicinalplants in several areas of the world besides China (Blanchan, 1922; Satyavati et al., 1987).Some physiological effects could undoubtedly be ascribed to a photosensitive component.In a folk prescription of a Hypericum extract as an antidepressant, there is a warning to faircomplexioned people about possible side effects of skin irritation (Moore, 1979). Certainspecies of Hypericum have a photosensitive effect when ingested by grazing animals and thepolycyclic quinone, hypericin, is considered to be responsible for this effect (Giese, 1980).There are about 200 species of Hypericum and the compound hypericin was first isolatedfrom the common European species Hypericum hirsutum L. It is a fluorescent red pigment.92The structure of the naphthaquinone system that forms the hypericin skeleton is shown withthe numbering of the carbons in Figure 4.1.Naphthabianthrone Skeleton of HypericinFigure 4.1: Hypericin skeleton with carbon positionsHypericin causes hemolysis of erythrocytes with exposure to light (Pace andMackinney, 1941). Hypericin has been shown to have antiviral activity against a number ofviruses. These include: radiation leukemia virus (Meruelo et al., 1988), Friend leukemiavirus, murine immunodeficiency virus (Lavie et al., 1989), equine infectious anemia virus(Kraus, et al., 1990), human immunodeficiency virus type 1 (HIV-1) (Schinazi et al., 1990),Moloney murine leukemia virus, influenza virus A, (Tang et al., 1990), vesicular stomatitisvirus, herpes simplex virus types 1 and 2, parainfluenza virus, and vaccinia virus (Andersenet al., 1991), Sindbis virus, murine cytomegalovirus (Hudson et al, 1991), humancytomegalovirus (Barnard et al., 1992), and duck hepatitis B virus (Moraleda et al., 1993). Inonly three of these studies was the role of light taken into account (Carpenter and Kraus,1991; Hudson et al., 1991; Moraleda et al, 1993).93Structure-activity studies elucidate the molecular geometry and chemical nature ofactive compounds that are necessary for activity or which affect the potencies. Chemicalderivatives, relatives and analogues of an active compound are tested and compared foractivity in order to discern the pertinent features. Several studies treated antiviral structure-activity relationships of hypericin and related quinonic compounds in the anthraquinone andanthrone groups. Pseudohypericin, which has a hydroxymethyl substitution instead of one ofthe methyl groups, shows diminished activity (Meruelo et al., 1988). By testing quinonescontaining parts of the hypericin ring system, Kraus et al. (1990) propose that the completering structure is required but is not sufficient for antiviral activity. Andersen et al. (1991)found activity against enveloped viruses with derivatives of emodin . Emodin can be thoughtof as the top portion of the hypericin ring system. The activity increases, though, withexpansion of the ring system to a bianthrone and to the full hypericin. Barnard et al.(1992)found differences in activity between anthrones and anthraquinones with different hydroxyl,methyl and carboxyl substitution patterns. Schinazi et al. (1990) tested anthraquinonessubstituted with hydroxyl, amino, halogen, carboxylic acid, substituted aromatic, andsulfonate groups against 1-11V-1. They found the polyphenolic and/or polysulfonatesubstituted ones to be most potent. The carbonyl function of quinones is considered to beessential to the antiretroviral activity of hypericin and analogues (Kraus et al., 1990; Lavie etal., 1990). The replacement of methyl side chains with more polar groups also results indiminished activity. The influence of light was never considered in these studies.In this study, three synthetic derivatives of hypericin and five bianthrones werescreened for light-mediated activity against the membrane enveloped Sindbis virus (SINV).Sindbis virus is sensitive to hypericin activity. This activity is shown when thevirus/compound mixture is exposed to fluorescent light prior to addition to cells (Hudson, etal., 1991, Lopez-Bazzochi et al., 1991). Natural compounds with similar structures were alsoassayed for activity against SINV and murine cytomegalovirus (MCMV).94Buckwheat, or Fagopyrum plants (Polygonaceae) have also been reported to causephotosensitive reactions when consumed by livestock. The compound fagopyrin has thesame naphthabianthrone central ring structure as hypericin (Thomson, 1971) but it has notbeen tested for antiviral activity. Three fungal compounds with a polyaromatic nucleus werealso tested in my studies. The perylenequinone cercosporin, first isolated from the soybeanblight, Cercospora kikuchii (Matsumoto et Tomoyasu) Gardner, has light-sensitive activityto mice and bacteria (Yamazaki et al., 1975). Its plant pathogenic effect is lipid peroxidationresulting in leaf membrane damage. It also has antifungal effects and has been shown toproduce both singlet oxygen and superoxide (Daub, 1987). Lavie et al. (1991) have reportedits antiretrovirus activity. Hypocrellin is another perylenequinone isolated from Hypocrellabambuase (B. et Br) sacc.. In their review of the hypocrellins Zhenjun and Lown (1990)discussed reports of its photosensitive antibacterial and antitumor effects. Cercosporin,hypocrellin A and fagopyrin are all deep red fluorescent pigments like hypericin. Duclauxinis a secondary metabolite in the Talaromyces (Frivad et al., 1990). It has also been isolatedfrom Penicillium stipitatum Thom (Kuhr and Fuska, 1973). It has a complex polycycliccentral skeleton that is dimerized from oxaphenalenones (Chexal et al., 1979).95Materials and MethodsChemicalsHypericin, three derivatives of hypericin, and five bianthrones were synthesized in thelaboratory of Dr. L.H. Zalkow at the Georgia Institute of Technology, Atlanta, Georgia(Gruszecka-Kowalik and Zalkow, 1991; Zembower, 1990). The chemical structures areshown in Figures 4.2 and 4.3.The crude extracts of Fagopyrum esculentum Moensch flowers and of Hypericumperforatum L. were provided by Z. Abramowski (Botany Dept. UBC). Dry residues weredissolved in ethanol for the bioassays. The structure of the compound fagopyrin found inFagopyrum is shown in Figure 4.4The compound duclauxin was provided by Dr. J. Jacyno (Microbial ProductsResearch Unit, South Atlantic Area Agricultural Research Center, U.S. Department ofAgriculture, Athens, Georgia). Cercosporin was provided by Dr. M. Daub.(Department. ofPlant Pathology, North Carolina State University, Raleigh). Hypocrellin A, hypocrellinperoxide and crude extract residue of Hypocrella bambuase were provided by Prof. J. Zhou(Phytochemistry Laboratory, Kunming Institute of Botany) The structures of thesecompounds are shown in Figures 4.5 -4.7.96Figure 4.2: Chemical Structure of Hypericin and Derivatives97Figure 4.3: Chemical Structure of BianthronesFigure 4.4: Chemical Structure of Fagopyrin98Figure 4.5: Chemical Structure of DuclauxinFigure 4.6: Chemical Structure of Cercosporin99Figure 4.7: Chemical Structures of Hypocrellin CompoundsAntiviral AssaysThe extracts or chemical compounds were assayed for inhibition of viral cytopathiceffects in serial dilutions following the same protocol described in the Material and Methodssection of Chapter II. One set of the compound/virus mixtures were exposed to 30 min ofvisible light before adding to the cell layers for infection. A parallel set was kept in the dark.For cytotoxicity assays, the compounds were exposed to 30 min of light. The adsorption timeon the cells take place in the dark in all the treatments. Experimental manipulations with thephotosensitive compounds were done in dim light. Light was provided by a bank of sixfluorescent tubes (General Electric F20/CW) at the incident energy of 1.5 W/cm2• The peakemission range of the fluorescent light was at 570-595 nm, and one of the major absorptionpeaks of hypericin is at 588 nm (Giese, 1980).Screening of hypericin analogues was done first against Sindbis virus. The activederivative was then tested against MCMV and poliovirus. Fagopyrum, cercosporin andduclauxin were tested against both enveloped viruses. The hypocrellin compounds wereassayed against MCMV.ResultsScreening of quinonoid compoundsThe antiviral activities of eight hypericin derivatives and related quinones bymicrotiter assay using 2-fold serial dilutions from 10 to 0.003 tg/ml, in comparison to that ofhypericin is shown in Table 4.1.100Of the three hypericin derivatives, compound EGK-149 showed significant activitywhich was in the same range as that of hypericin. It was then tested against MCMV and wasactive in the plaque assay at 1.25 pg/ml in light.Fagopyrum AssaysThe crude extracts of Hypericum and Fagopyrum were assayed in 2-fold serialdilutions starting from the highest concentration of 100 lig/ml. The results are shown inTable 4.2.Fungal Compounds AssaysIn 2-fold serial dilution assays starting from the highest concentration of 100 1.1g/ml,the compound duclauxin showed no activity. The activity of cercosporin is shown in Table4.3 Cercosporin showed a cytotoxic effect beginning at 5 lig/m1 and higher concentrations.At the concentration effective in the dark against SINV, the cells appeared abnormal eventhough there was no virus infection.The activity of the hypocrellin compounds are shown in Table 4.4. The Hypocrellaextract and hypocrellin A both showed light-enhanced activity but the endoperoxide ofhypocrellin did not. However, hypocrellin A began to show cytotoxicity at a concentration of10 pi/mi.101Compound112111=11EGK-138EGK-149EGK-162DZ-IV-48DZ-IV-54DZ-IV-55DZ-IV-64DZ-IV-65Concentration in pg/m1Light Dark0.02 0. 105.0 10.00.02 0.255.0 N.A.N.A. N.A.N.A. N.A.* 5.0 * 5.0N.A. N.A.N.A. N.A.Minimum Active Concentration, mlTable 4.1Minimum Active Concentrations of Hypercin CompoundsAgainst Sindbis VirusN.A. = no activity* = no viral infection but visible cell effectTable 4.2Minimum Active Antiviral Concentrations of Hypericum and Fagopyrum Crude Extracts102Table 4.3Minimum Active Antiviral Concentration of Cercosporin^^^^^`\^Active ConcentrationVirus'^DarkSINV^0.3^6.0MCMV^2.5 10** No viral infection but showing cytotoxic effects.Table 4.4Minimum Active Antiviral Concentrations of Hypocrella CompoundsMinimum Active Concentrations in . / ml* No viral infection but showing cytotoxic effects.(Assays for SINV not interpretable due to loss of infectivity in virus stock)103DiscussionIn the screening of hypericin relatives and similar compounds, light-mediatedantiviral activity was found in the crude extract of the buckwheat plant Fagopyrum, and fourof the synthetic hypericin analogues. The Fagopyrum extract was active in a range ofconcentrations similar to the Hypericum extract. Hypericin has been shown to be aphotosensitive antiviral compound from Hypericum. Since the two plants have a similarhistory of causing photosensitive reactions, and Fagopyrum contains the compoundfagopyrin which is structurally similar to hypericin, it is reasonable to attribute the antiviraleffect of Fagopyrum to fagopyrin. Fagopyrin differs from hypericin only in the substitutiongroups at positions 3 and 4. This would suggest that the necessary structure for activity is thecentral naphthabianthrone ring system, or some portion of it. Lavie et al. (1990) found thatthe elimination of the carbonyl functions on the ring system greatly reduces reversetranscriptase inhibition.The three synthetic hypericin derivatives (EGK compounds) tested, all differed fromhypericin in only the non-hydroxyl substitution groups on the naphthabianthrone center. Thethree derivatives showed different degrees of potency. The compound EGK-149, 2,5,9,12-tetra(carboxyethylthiomethyl) hypericin, showed significant antiviral activity with similarpotency to that of hypericin. Kraus et al. (1990) suggested that the hydroxyl groups may beimportant in antiretroviral activity, because the naphthabianthrone system alone is notsufficient for activity against equine infectious anemia virus (EIAV). The lesser potency ofthe two other derivatives found in this present study indicated that the activity from thepresence of both of those features could still be greatly influenced by other substitutiongroups. Substitution groups of EGK-149 contain sulfur. Schinazi et al. (1990) foundanthraquinones with sulfonate substitutions to be among the more active of an array testedagainst HIV-1, even though the potencies are much less than that of hypericin. The role of104sulfur in the molecule would be an interesting area for further structure-activityinvestigations. The nature of action of EGK-149 is the subject of Chapter V.Compound EGK-138 is hypericin dicarboxylic acid. It had been found to beineffective in reducing the production of radiation leukemia virus (RadLV) in mice by Lavieet al. (1990). They associate the reduction in activity to the increase in polarity of side chainsat positions 3 and 4. Analogues with acetoxyl (hypericin diacetate) and hydroxyl (hydroxy-desmethylhypericin) groups at those positions also have diminished activity. Desmethylhypericin, lacking the 2 methyl groups, has good activity in the direct inactivation of virionsbut is ineffective in in vivo assay. Meruelo et al. (1991) proposed that the methyl groupsaffect the retention of the molecule by cells. Compound EGK-162 is a glucoside that was notcompletely characterized. It is a green pigment, unlike the intense red of hypericin and EGK-149. Its different absorption spectrum may be one of the reasons for its lesser activity shownin the assay conditions.While comments on the activity of fagopyrin should be made from studies made withthe purified compound, it is interesting to note that fagopyrin and EGK-149 both have largeside chains on the hypericin ring system. EGK-149 , especially, has these groups at fourpositions that are not substituted in hypericin. All active hypericin compounds reported thusfar have hydroxyl groups at carbons 1,6,8 and 13, as well as at 3 and 4 (or 10 and 11). Howthese substitution groups relate to the other molecular elements of hypericin deemedimportant for action remains to be determined.The assay of the bianthrones relates to the effect of the nature of the central ringskeleton on activity. Bianthrones do not have the two central (naphtha) rings of the hypericinskeleton. Only one of the bianthrones assayed, DZ-IV-55 was active. Bianthrone with noside chains is not effective in inactivation of RadLV (Lavie et al., 1990). Emodin bianthrone,105however, showed activity against five viruses (vaccinia virus, parainfluenza, herpes simplexvirus types 1 and 2, and vesicular stomatitis virus) in a study comparing the activity ofseveral anthraquinones (Andersen et al., 1991). The activity is not as great as that ofhypericin but the D,L- diastereoisomers of emodin bianthrone are consistently andsignificantly more active than the meso -isomer. This difference is attributed to the closerresemblance in three dimensional structure of the planar extended ring of the D,L-isomer tothe hypericin ring, whereas the meso -isomer ring is more bent across the molecule betweenthe two anthrone groups. Compound DZ-IV-55 in this study is an emodin bianthrone, but itis not known whether it is a mixture of isomers. It showed some activity, but the activeconcentrations caused an alteration to the assay cells even though they remained viable.Looking at the differences in substitution groups on the bianthrones tested, DZ-IV-55 has thesame pattern as that of hypericin. This corroborates the interpretation that the substitutiongroups at positions 3,4 and 9,10 are significant in the structure-activity relationship.Hypericin (8-ring) can be synthesized via emodin bianthrone (6-ring) from themonomer, emodin (3-ring), as depicted in Andersen et al. (1990). Emodin or emodinanthrone can be considered to be one of the anthrone portions of the hypericin skeleton.Andersen et al. (1991) found aloe emodin to be inactive against six viruses tested, includingherpes simplex virus (HSV) types 1 and 2. Sydiskis et al. (1991) found it to inactivate HSV-1 and -2, as well as three others: varicella-zoster virus, pseudorabies virus and influenzavirus. Both groups found it to be inactive against rhinovirus. Carpenter and Kraus (1991)found emodin to be inactive against EIAV, even with exposure to light. Both emodin andanthrone are inactive against RadLV (Lavie et al., 1990).The differences in reported results is a reminder that there are many elements that canmake comparisons between antiviral evaluations challenging. Assays can be done using anumber of very different methods (as discussed in Chapter 2), and assays may be conducted106with a variety of viruses, and viruses may differ between strains. Stereochemistry of antiviralcompounds has to be considered, and the influence of light must be incorporated into thetesting strategy.The structures of cercosporin and hypocrellin are similar to hypericin, but representan extended quinone originating from naphthalene. They have in common the central 5-ringperylenequinone skeleton and two methoxyl substitution groups (C2, Cii). Cercosporin has amethylene-dioxy bridge between C6 and C7, and two alcohol side chains (Ci, C12). It didshow light-mediated activity against membrane enveloped viruses in this assay, corroboratingits previously reported light- and oxygen-mediated activity on membranes. Hypocrellin Ahas a different substitution pattern with a 7-membered ring formed between Ci and Ci2 andmethoxyl groups at C6 and C7. It showed more potent light-mediated activity thancercosporin, but it showed cytotoxicity at concentrations higher than 10 lig/ml. Theendoperoxide of hypocrellin was only marginally active. This inactivity could be explainedby the fact that endoperoxides are often the adduct products of a singlet oxygen reaction andwould probably not produce another photodynamic effect. It is possible for a photo-activereaction to result in an active end product. If this is not the case, it indicates that the activityoccurred during the short-lived excited singlet oxygen generation state. The requirement foroxygen for the photo-action of cercosporin has been demonstrated (Yamazaki et al., 1975). Itappears that the original configuration of the center ring where the peroxide formed wasimportant in generating the activity. The formation of the peroxide function disrupts thearomatic extension between the two anthraquinone groups. It was shown in the studies ofanthraquinones by Andersen et al. (1991), that antiviral activity increased with the expansionof the ring system from emodin to bianthrone to hypericin. It is likely that the samereduction in the number of extended aromatic rings would result in a loss of activity. Theelectrons in the p-orbitals of conjugated bonds extending over several ring systems are easilyexcited by visible light (Hader and Tevini, 1987) and would generate the photo-activity. The107structure-activity relationships of these compounds with perylenequinone skeletonscorroborates with the conclusion that the extended quinone central skeleton is important inphotodynamic reactions. In a screening for protein kinase C inhibitors, the perylenequinonecalphostin C and hypericin and pseudohypericin were found to be active (Takahashi et al.,1989). The quinone skeleton was considered to be important in this similarity even thoughthe role of light was not taken into account.The importance of the extended quinone skeleton to photo-activity is also supportedby the fact that duclauxin did not show any activity. It has a different type of polycyclicskeleton even though carbonyl functions are present and excited triplet state carbonyl groupscan play a role in the transfer of energy in the promotion of a molecule like oxygen to asinglet state (Cilento, 1980). The oxaphenalenone-derived skeleton of duclauxin does nothave the same degree of extension of aromatic rings as hypericin and the perylenequinones.In summary, the structural characteristics significant to the activity of hypericinappear to be: a) the extended quinone central skeleton, b) the quinone carbonyl functions, andc) hydroxyl substitution groups at certain positions. These features are necessary for activitybut the activity can be affected by other substitution groups as well.108Chapter IV ReferencesAltamirano, M., Hudson, J.B., and Towers, G.H.N. (1986) Induction of cross-links in viralDNA by naturally occurring photosensitizers. Photochem. Photobiol. 44, 187-192.Andersen, D.O., Weber, N.D. Wood, S.G., Hughes, B.G., Murray, B.K. and North J.A.(1991) In vitro virucidal activity of selected anthraquinones and anthraquinonederivatives. Antiviral Res. 16, 185-196.Blanchan, N. (1922) Wild Flowers Worth Knowing. 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Chem. 39(1),287-288.Zembower, D.E. (1990) The synthesis and structure-activity relationships of biologicallyactive anthraquinones. Ph.D. thesis, Georgia Institute of Technology, Atlanta.Zhenjun, D. and Lown, J.W. (1990) Hypocrellins and their use in photosensitization.Photochem. Photobiol. 52, 609-616.112Chapter VNature of Antiviral Action of the Hypericin Derivative EGK-149IntroductionThe new synthetic derivative of hypercin, 2,5,9,12-tetra (carboxyethylthiomethyl)hypericin (EGK-149) showed potent photosensitive antiviral activity in the screening ofhypericin analogues. The nature of its action was compared with that of hypericin. Toelucidate the nature of action of a compound, one has to consider the range of potential targetsites for the action. The possible sites of action for an antiviral agent in the viral infectioncycle are: a) direct inactivation of virion, b) attachment to and entry of host cell, c) uncoatingof virus genome, d) viral protein synthesis transcription and translation, e) replication ofgenome, and f) assembly and release of progeny virus. The functions that are potentialtargets at each one of these stages will be briefly described, with examples of knowninhibitors and known or proposed mechanisms of actions.Direct inactivation of the virus particle takes place extracellularly. Many compoundssuch as acids, urea, phenolics, and detergents can have such virucidal effects but they are notconsidered therapeutically useful because of their cytotoxicity (Vanden Berghe et al., 1985).Compounds such as hypericin and fluoranthene are considered virucidal because they cancause a reduction in viral infectivity with treatment of the virus prior to addition to the cell.This was demonstrated in the treatment exposing only the virus / compound mixture to UVAprior to cellular infection. However, the fact that an antiviral effect is seen with a pre-infection treatment of the virus does not necessarily mean that the virus was rendered non-infectious. The effect can still be due to inactivation of a viral component that is part of a113latter stage of replication. The therapeutic potential of an extracellular virucidal effect wouldbe in the prevention of the spread of infection.One of the factors that results in the restriction of many viruses to specific hosts is therequirement of specific receptors on the cell surface for recognition and attachment. Theantiviral activity of sulfated polysaccharides such as dextran sulfate has been attributed toinhibition of the binding of HIV to its cellular receptor (Mitsuya et al., 1988). Other forcessuch as electrostatic interactions, hydrogen and hydrophobic bondings are also thought toinfluence the viral binding (Lonberg-Holm, 1980). Polyionic compounds have been shownto inhibit Sindbis virus binding to cellular receptors. Polyanions appear to act on the virusparticles while polycations bind to the cell membrane receptors (Mastromarino et al., 1991)The penetration of the cell by the virus is thought to take place via one of the cellularendocytic processes like receptor-mediated endocytosis, or direct translocation with non-enveloped viruses (Dimmock, 1982). With enveloped viruses, fusion occurs between the celland envelope membranes. The fusion of paramyxovirus has been shown to be inhibited bysynthetic peptides (Richardson and Choppin, 1983).The process of uncoating releases the viral genome from the protein capsid. Itappears that the process can involve multiple stages and that greater time is required for therelease of nucleic acid from more complex viruses (Matthews, 1973). The capsid structure ofsome picornaviruses has been determined to the three-dimensional atomic level, and containshydrophobic pockets in one protein located within the 'canyon' depressions on the capsidsurface. Compounds synthesized by the Sterling-Winthrop Research Institute have beenshown to prevent the uncoating of rhinoviruses by binding to these hydrophobic pockets(Smith et al., 1986). These 'WIN' compounds consist of isoxazole and oxazolinylphenoxylgroups linked by aliphatic chains. It is thought that the mechanism of action is the inhibitionof capsid dissembly through decreasing the flexibility of the protein. The 'canyon114hydrophobic pocket' structure is found in the icosahedral capsid proteins of a number of plantand animal viruses. As this structure is generally lacking in cell proteins, it has beenproposed that this is a target site for which selective antiviral agents could be designed to beapplied to viruses with this feature (Rossman, 1989). .Virus replication involves the synthesis of viral proteins and nucleic acids. Themajoritiy of antiviral agents approved for clinical use, such as idoxyuridine, acyclovir (ACV)and azidothymidine (AZT), are nucleoside analogues that interfere with these replicationprocesses. Such analogues can act through being incorporated in a genetic process bymimicking the normal nucleosides but in turn disrupt the normal function (Montgomery,1989). The drug ACV inhibits virus induced DNA polymerase activity (Furman et al, 1979),and AZT triphosphate inhibits reverse transcriptase (Furman et al, 1986). Guanidine, 3-methyl quercetin, and the methoxyflavone isolated from a Chinese medicinal plant have allbeen shown to inhibit RNA synthesis (Tershak, 1982; Castrillo and Carrasco, 1987; Ishitsukaet al., 1982). The methoxyflavone does not directly inactivate the RNA polymerase complexbut appears to prevent its formation in the infected cell. Polynucleotides have been shown toinhibit influenza viral transcriptase (Smith et al., 1980), and the nucleoside ribavirintriphosphate inhibits influenza RNA polymerase (Eriksson et al., 1977). The molecularapproach to interference with genetic expression has also been taken. Oligonucleotidesantisense to messenger RNAs (mRNA) have been shown to inhibit viral protein synthesis(Smith et al., 1986; Lemaitre et al., 1987). Sim (1990) summarized the current knowledge ofvirus-specific inhibitors and discussed the future potential of using the molecular approach inthe discovery of antiviral agents. It is proposed that the identification of virus-coded proteinsnecessary in viral replication will indicate targets for the selection and design of antiviralagents that do not impede host functions.115Viruses produce structural and non-structural functional proteins such as polymerases.In many viruses, proteins are synthesized as large precursors that are then cleaved byproteases. Protease inhibitors have been shown to suppress the production of infectiousinfluenza virus but the enzymes affected could be of cell origin (Zhirnov et al., 1982). Thevirus envelope is composed of cell membrane components and viral glycoproteins. Anumber of antiviral compounds, like tunicamycin and the plant alkaloid castanospermine, caninhibit glycosylation and exhibit antiviral effects (Klenk and Schwartz, 1982; Walker et al.,1987). The assembly of virus particles is considered to be a generally self-regulated processwhere the capsid is constructed from basic subunits but the genome also has to be packagedwithin the capsid to produce an infectious virion. The progeny assembly and releaseprocesses are not very well understood.A summary of the different sites of antiviral action should also include the activity ofinterferon which is a cellular product and not virus-specific. A virus infected cell isstimulated to synthesize and export interferon. Interferon signals other cells to modulate cellfunctions in a protective manner and to produce an antiviral protein. It is thought that theprotein impedes the transcription of viral mRNA (Jackson, 1986). Interferon has been usedin treatment to induce this effect. The extract of Melia azedarach L. has also shown theability to induce an antiviral state (Wachsman et al., 1987). The nature of the effect of theisolated glycopeptide meliacin resembles that of interferon but it does not appear to beinterferon-mediated (Andrei et al. 1988). The mechanism of this meliacin protective effectand the role of this protein in the plant remain to be elucidated.The scope of the potential sites of antiviral activities in the virus infection cycle iswide and far from being fully understood or explored. For most antiviral compounds, themechanisms of action have not yet been elucidated in full detail. Proceeding from what isknown and proposed about the nature of the activity of hypericin, the mechanism of action of116its active derivative EGK-149 was investigated. The activity of hypericin has been examinedin a number of studies and there is probably more than one mechanism involved. Hypericinwas reported to have antiretroviral activity by direct inactivation or interference with theassembly and release process (Meruelo et al., 1988). The study also showed suppression ofreverse transcriptase (RT) activity in infected cell cultures. Lavie et al. (1989) documentedabnormal viral cores using electron microscopy. It is suggested from comparison withreports concerning other viral core formations that hypericin may disrupt the polyprotein orproteins essential in the assembly process, and infectivity is lost when the RNA is notincorporated into the progeny particles. The suppression of RT activity does not take placewhen tested with purfied enzymes.Hypericin is active against several retroviruses, including the immunodeficiency andleukemia viruses (listed in Chapter IV) . Hypericin also inhibits the activity of the enzymeprotein kinase C (Takahashi et al., 1989). Phosphorylation activity is part of the reversetranscription process and its inhibition could result in the inhibition of reverse transcriptaseactivity reported by Meruelo et al. (1988) that is not from direct inactivation of RT. Schinaziet al. (1990), however, showed hypericin inhibition of the HIV-1 RT, even though this effectcan be negated with the addition of bovine serum albumin (BSA). They suggested that thiseffect of hypericin on the enzyme may be from a non-specific binding to proteins. Carpenterand Kraus (1991) showed the complete inactivation of equine infectious anemia virus reversetranscriptase activity by hypericin with 60 minutes of exposure to light. Viral infectivity,however, can be eliminated with only 10 minutes of exposure to light indicating that RTinhibition is not the only mechanism of action. Hudson et al. (1991) reported the light-enhanced inhibition of MCMV occurring at an early stage of the replication cycle and likelyfrom action on an intracellular target. Moraleda et al. (1993) proposed that the activity ofhypericin against duck hepatitis B virus (DHBV) takes place in morphogenesis steps late inthe replication process.117Tang et al. (1990) demonstrated that hypericin is active against membrane envelopedviruses but not against nonenveloped viruses. Exposure of the compound and virus to lightgreatly enhanced the antiviral effects against enveloped viruses, even though there is still asignificant effect in the dark (Lopez-Bazzochi et al., 1990; Hudson et al., 1991). Meruelo etal. (1988) found that the compound was localized in the surface membrane when incubatedwith cells. These findings about hypericin indicate that the viral membrane is a site for thephotodynamic mechanism of antiviral action. These studies show that there is probably not asimple explanation for the mechanism of action(s) of hypericin. The role of light was alsonot considered in all of these studies. While hypericin has been shown to have activity in thedark, its photosensitivity makes it important to control light conditions in studies to elucidatethe mechanism of action.A probable mechanism for action on membranes is via singlet oxygen produced bythe promotion of hypericin to a reactive state by the absorption of photons. It has beenshown that hypericin does generate singlet oxygen in visible light (Duran and Song, 1986).To confirm that the membrane was the likely site of action, hypericin and EGK-149 weretested against a nonenveloped virus (polio) and another enveloped virus (MCMV). Since thepossibility of other mechanisms of action that may be independent of light have beenproposed in previous studies, different stages of the virus infection cycle were treated withand without light to better elucidate the site of activity.The singlet oxygen generated by photosensitized hypericin can react in two ways withother molecules. It can form a new product with them such as in the formation ofendoperoxide compounds, or it can transfer its energy to excite another molecule returning tothe ground state itself. This phenomenon is called quenching (Hader and Tevini, 1987).Singlet oxygen commonly reacts with conjugated double bonds, aromatics and heterocyclesand many such bonds are found in biological molecules. For example, lipid peroxidation is118the formation of hydroperoxides with the fatty acid side chains of membrane lipids thatcauses membrane damage. Well known quencher molecules include: the I3-carotenes, a-tocopherol (vitamin E), phenols and azide compounds (Halliwell and Gutteridge, 1989). Acompound with a high rate of reaction with singlet oxygen can be considerd to be a 'singletoxygen scavenger'. To test if singlet oxygen is involved in a photodynamic reaction, aquencher or singlet oxygen scavenger compound can be added to the system to see if theeffect is reduced or eliminated (Foote, 1987).To investigate if there is indeed a singlet oxygen effect in the antiviral activity ofcompound EGK-149, it and hypericin were assayed in the presence of cholesterol as a singletoxygen scavenger. Cholesterol has been used as a trap for singlet oxygen because the twomolecules react to form 313-hydroxy-5a- cholest-6-ene-hydroperoxide (Kulig and Smith,1973). The viral membrane of the Sindbis virus used in the assay is a lipid bilayer derivedfrom the host plasma membrane with a representative sample of cellular membrane lipids andcholesterol is a known component (Lenard, 1980). Adding cholesterol to the assay thereforewould not raise the question of toxicity as would the use of some other effective quencherslike sodium azide.The molecular target of the singlet oxygen photodynamc effect can be part of themembrane system. Damage to membrane liposomes by photosensitizers has beendemonstrated (McRae et al., 1985). The viral membrane is composed of a host-derived lipidbilayer and viral coded membrane proteins (Marsh, 1987). The host membrane proteins areexcluded and the viral envelope spike glycoproteins are involved in the receptor binding andfusion processes of host cell entry. When the membrane is the proposed site ofphotodynamic reactions, both lipid components and the proteins are possible targets ofaction. The structure of the Sindbis virus particle has been well-characterized. The viralRNA is packaged in an icosahedral nucleocapsid composed of one 30 kilodalton (kD) capsid119protein (Fuller, 1987). The surrounding envelope has two glycoproteins that span andprotrude from the lipid bilayer; they are arranged in trimers in an icosahedral lattice(Harrison, 1986). The spike proteins, designated El and E2, have their major domainsexternal to the membrane (Schlesinger and Schlesinger, 1986). It was hypothesized in thepresent study that the protein domains exterior of the envelope are potential targets ofphotodynamic action. Schinazi et al. (1990) described a non-specific binding to proteinsshown by hypericin. The photosensitive compound oc-terthienyl was reported to cause across-linking of membrane proteins in bacteria in UVA (Downum, et al., 1982). It also haspotent antiviral activity (Hudson et al., 1986). Moraleda et al. (1993) observed that hypericincaused a cross-linking of the preS envelope protein of DHBV surface antigen particles andinferred that it also occurs with virus particles. To see if the antiviral action could be takingplace on the viral proteins, the proteins from Sindbis virus particles treated with hypericin,EGK-149, and oc-terthienyl, in light and dark, were separated by gel electrophoresis in orderto discern any alterations.Material and MethodsTime of treatment antiviral assaysAntiviral assays with Sindbis virus were done using the plaque reduction method(described in Material and Methods of Chapter II). The virus / cell system was treated withhypericin and compound EGK-149 , and exposed to light at different periods in relation tothe virus infection process. The treatments consisted of the following stages, with a paralleltreatment done in the dark:1201)cell layer only treated for 1 hr and compound removed prior to virus adsorption,2) virus particles only for 30 min, compound remained during adsorption in the dark,3) virus particle only for 30 min, compound removed before the adsorption period inthe dark,4) virus and cell during 1 hr of adsorption,5) cell layers post-adsorption, at 0-1 hr, 3-4 hr and 5-6 hr.The incident energy of the fluorescent light source was 1.5 W/cm 2 . The exposure to lighttook place in the Environ-Shaker cabinet, at 40C with virus particles only and at 37 0C withcells and virus. For exposing the virus particles only to the compounds (as in treatment 3),the virus suspensions of 1000 pfu/ml were treated with the compounds for 30 min in light ordark. The suspensions were concentrated in Centricon-30 miniconcentrators (Amicon) whichhave filters with a 30,000 molecular weight (MW) cutoff by centrifuging for 10 min at 5000G in a Sorval SS-34 rotor at 40C. The particles were washed twice this way with PBS andthen resuspended in culture medium for assay.Time of treatment assays were limited with MCMV. The use of the murine cell line(3T3-L1) that was necessary for its assay was discontinued due to observable changes inmonolayer properties.Singlet oxygen mechanism assaysCholesterol (Sigma C3137) was dissolved in 95% ethanol and added to SINV plaquereduction assays in light and dark as a singlet oxygen scavenger at concentrations between0.1 and 100 µg/ml. The compound / virus mixture solution was essentially saturated withcholesterol at 50 fig/ml. In addition to cell only and virus only controls, virus was treatedwith the same concentrations of cholesterol only.121Viral protein separationSindbis virus particles at 5 x 10 5 pfu/ml were treated with one of the threecompounds, EGK-149, hypericin or a-terthienyl for 30 min in light or dark. The compoundconcentrations tested were: 0.1, 1.0, 10 and 100 ps/ml. The suspension was added to equalvolume of 20% polyethylene glycol (PEG) and the proteins were precipitated in thecentrifuge (Sorval) by spinning at 15,000 G for 2 h. The pellet was resuspended in 1 ml ofdistilled water and respun in a microcentrifuge (Eppendorf 5415C) for 20 min at 12,000 G.The proteins were separated by 10% sodium dodecylsulfate protein agar gel electrophoresis(SDS-Page). Proteins were quantified with the Bradford Protein Quantification Assay and 25or 50 tg were loaded per lane to gels on a vertical slab gel unit (Mighty Small SE 200,Hoefer Scientific). Control lanes were run simultaneously with untreated virus proteins.Since the virus stock was purified from cell cultures, controls were also run with 3T3-L1 cellproteins. The gels were stained with Coomassie Blue. The molecular weights (MW) of theproteins were calculated in reference to standard markers (Sigma MW SDS-70L Kit). TheMW of the envelope proteins are 50 kD for El and 60 kD for PEI The capsid protein has aMW of 30 kD (Bonatti et al., 1979).ResultsTime of Treatment Activity AssaysPlates with the fixed and stained cell layers of a Sindbis virus plaque reduction assaywhere the cell / virus system were treated at different times of the infection process, in lightand dark, are shown in Figure 5.1. The percentages of plaque reduction are listed in Table5.1. The effects of EGK-149 against MCMV are shown in Table 5.2.122Figure 5.1: Sindbis Virus Antiviral Plaque Reduction Assay Platesfrom Treatments with Compounds at Different Stages of Viral Infection Cycle.123CompoundTable 5.1: Percentage of Sindbis Virus Plaque Reduction From Treatments at Different Stages of Viral Infection CyclePercentage of Sindbis Virus Plaque Reduction from Different Times of TreatmentStage_ of Infection Process Treated with CompoundA) Pre-InfectionB) VirusOnlyC) Virus andInfectionD) InfectionTime OnlyE) Post Infection0-1 Hr 3-4 Hr 5-6 Hr0 100 100 43 0 0 00 100 81 79 0 0 00 100 100 79 0 0 00 62 62 0 0 0 00 0 0 2.5 0 0 00 51 56 0 0 0 0Stage of treatment with compound in light and dark:A) cell layer only for 1 hr and compound removed prior to virus addition.B) virus only for 30 min (compound remained during infection in the dark).C) virus only for 30 min and with cell layer during 1 hr of infection.D) virus and cell layer during 1 hr of infection.E) cell layer only post-infection after removal of virus.0 % = no antiviral effect^100 % = total elimination of viral infectionLightDarkHypericin0.05 pg/m1EGK-1490.05 pg/m1Hypericin0.05 pg/mlEGK-1490.05 pg/mlEGK-1490.1 pg/mlEGK-1490.1 pg/m1124Table 5.2Percentage of Murine Cytomegalovirus Plaque Reduction from Treatment with Compound EGK-149 at Different Stages of Viral Infection CyclePercentage of Plaque Reduction from Different Times of TreatmentStage of Infection Process Treated with Compound *A) Pre-infection B) Virus Only C) Virus and Infection D) Infection OnlyLight 0 0 88 100Dark 0 0 0 15Concentration of compound EGK-149 = 0.1 µg/ml.* same as defined in Table 5.1.0 = no antiviral effect^100 % = total elimination of viral infectionThe virus particles that were treated with the compounds and recovered after washingbefore adding to cells were not infective.Singlet oxygen mechanism assaysThe effects of the presence of cholesterol as a singlet oxygen scavenger on theantiviral activity of hypericin and EGK-149 are shown in Figures 5.2 and 5.3.Effect of hypericin and EGK-149 on Sindbis virus proteinsThe structural proteins of Sindbis virus are indicated on the SDS-Page gel shown inFigure 5.4. The 30 kD capsid protein showed a retardation shift when the virus particles havebeen treated with 10 or 100 pg/ml of EGK-149 in light. No other protein alterations wereseen, with the EGK-149 treatments in dark or with the hypericin and a-terthienyl treatmentsin light or in dark.125Figure 5.2: Percentage of Sindbis Virus Plaque Reduction by Compound EGK-149 in the Presence of CholesterolPercentagePlaqueReduction100 -80-60-40-20-00^0.1^1.0^10^50^100Concentration of cholesterol lig/m1o = cholesterol control, in light and dark= EGK-149 0.05 pg/ml, in light^* = EGK-149 0.05^in dark^A = EGK-149 0.10 p.g/ml. in light^• = EGK-149 0.10 n.ml, in darkEach symbol point represents the result from one assay.126•Figure 5.3: Percentage of Sindbis Virus Plaque Reduction by Hypericin in the Presence of CholesterolPercentagePlaqueReduction100-80-60-40-20-00^0.1^1 .0^10^50^100Concentration of cholesterol ..t,g/m1o = cholesterol control, in light and darkA = hypericin 0.05 pg/ml, in light^• hypericin 0.05 pg/ml, in dark0 = hypericin 0.10^in light # = hypericin 0.10 tg/ml, in darkEach symbol point represents the result from one assay.127Electrophoretic Gel of Sindbis Virus Proteins•••■••••Lane: 1 2 3 4 5 6MW L LSW. EGK-V^--- Hypericin149D I) L D I. D87Lane 1: Untreated virusLanes 2, 3: EGK-149, 100 pg/ml.Lanes 4, 5: Hypericin, 100 pg/ml.Lanes 6, 7: Hypericin, 10 pg/ml.Lane 8: Hypericin, 1 pgiml.C = capsid protein^E = envelope proteinsFigure 5.4: Electrophoretic Gel (SDS-PAGE) of Sindbis Virus ProteinsTreated with Compound EGK-149 and Hypericin.L = treatment in light D = treatment in darkCapsid protein in lane 2 showed retarding shift.128DiscussionTo determine the site of antiviral action of EGK-149, the virus / cell system wastreated with the compound and with hypericin at different times which encompass differentstages of the virus infection cycle. It has been shown that the hypericin has an extracellularvirucidal activity against enveloped viruses. This action appears to be from a light-mediatedsinglet oxygen effect on the virus membrane. The novel hypericin derivative EGK-149 hasalso exhibited this light-mediated virucidal activity. The time of treatment assays were alsoto compare the actions of the two compounds. The action of hypericin appears to be complexand likely entails more than one mechanism. Comparison of the nature of action with aderivative would continue the studies to elucidate and distinguish these mechanisms.In addition to the light-mediated effect on membranes, hypericin has also exhibitedsignificant activity in the dark (Lopez-Bazzochi et al., 1990). It inhibits the activities ofmonoamine oxidase (Suzuki et al., 1984) and protein kinase C (Takahashi et al., 1989) inenzyme assays; and the release of reverse transcriptase in infected cells (Meruelo et al.,1988). Sites of actions in the intracellular viral replication process have been proposed for itsactivity. Lavie et al. (1989) found disruption of the assembly of radiation leukemia progenyvirions. This event would take place late in the replication process at some time after the cellhas been exposed to the virus. Hudson et al. (1991) suggested the possiblity of interferencein MCMV transcriptional events early in the replication cycle. In the treatment of the cellsafter exposure to SINV with hypericin or EGK-149, there was no reduction in plaqueformation. This indicates that there was no interference with the intracelluar replicationevents with SINV. It is unfortunate that time of treatment assays were only conducted withEGK-149 against MCMV for technical reasons and there were no post-infection treatmentassays. There appeared to be an inhibition effect with the treatment of the cell and virusduring the adsorption time when the virus infects the cell. Since the cells were exposed to129virus for 1 hour, it is possible that the compound was acting on early intracellular eventsbesides the membrane disruption. The action of hypercin and EGK-149 on the earlyintracellular events of MCMV infection definitely needs further investigation. It had shownvirucidal activity at the higher concentration of 1.25 µg/m1 in light (Results, Chapter IV)where the cells were not exposed to light. That concentration in light would be cytotoxic aswell.Neither of the compounds exhibited any pre-infection treatment antiviral effects. Ininhibition of viral cytopathic effects assays with serial dilutions, hypericin showedapproximately twice the potency as EGK-149 in the dark (see Table 4.1, Chapter IV). Thisdifference is seen again in effects of treating SINV particles only and treating both the virusand the cell during infection. In the dark, hypericin at 0.05 tg/m1 caused 62 % plaquereduction as compared to 51 and 56 % by EGK-149 at 0.1 µg/ml. It should be noted that inmany reports concerning the activity of hypericin, the concentrations used (greater than 1pg/m1 ) would probably be cytotoxic if cells were used in assays and were exposed to light inthe presence of the compound. Light-induced cytotoxicity would not affect assays of purifiedenzyme activity or direct virion inactivation, but as the role of light is important in theactivity of hypericin compounds it should be taken into consideration in all studies.Inhibition of infection was complete with treatment of the virus in light. The extentof inhibition was reduced when the virus was not treated prior to adding to the cell layer. Inthis treatment where the virus and compound were added simultaneously to the cell layer,there was essentially no activity in the dark. This suggests that with SINV the action ofhypericin and EGK 149 was directly against the virus particle. This result is in accordancewith the report by Tang et al. (1990) of the virucidal activity of hypericin against envelopedviruses. Since the compounds remained with the virus suspension during the infection time,even though it was in the dark it was not certain that the site of action was limited to the virus130particle. It would be interesting to see if the infectivity is the same if only the virus wasexposed to the compounds. The procedure to remove the compounds from the virussuspension before addition to the cells, however, was not developed completely. There wasno infectivity from the virus after removal of the compounds but there was also only marginalinfectivity from the control cells, showing that there was damage to the virus from themanipulations. I believe it is possible to refine the protocol to accomplish this assay. Theloss of infectivity with exposure of only the virus to the compound would confirm theindication from the present evidence that the antiviral effect is from the direct inactivation ofthe virus.The effect in the dark from the prior treatment of the virus indicates that the activity islight-enhanced but not entirely light-dependent. The photosensitive activity is attributed tothe generation of singlet oxygen which damages the membrane. It is likely that the singletoxygen effect took place much faster than the dark effect. Longer time requirement for theoccurrence of the dark effect could explain why there was no activity in the dark shownwithout prior treatment of the virus particles. The effect in the dark could be independent ofoxygen but this has not been demonstrated. The membrane envelope is a feature shared bymany groups of viruses, but viruses also differ greatly in other aspects of molecularcomposition. It is reasonable to conclude that hypericin compounds have a light-enhancedsinglet oxygen type effect against membrane-enveloped viruses, but there is evidence foractions interfering with intracellular replicative events of specific virus groups.Addition of cholesterol as a singlet oxygen scavenger was used to see if singletoxygen participates in the photodynamic reaction causing the antiviral effect. A highconcentration of a scavenger is expected to inhibit a singlet oxygen dependent reaction. Theantiviral effect of EGK-149 in light was reduced in a dose-dependent manner by the presenceof cholesterol (Figure 5.2). This indicates that singlet oxygen is involved in the reaction.131The effect was not as apparent with hypercin (Figure 5.3) at the concentrations tested.Hypericin has a stronger effect in the dark than EGK-149 and this is evident from thereduction in viral plaque formation caused by hypericin in the dark at the same concentration.While there was a slight plaque reduction effect, the dark effect appears to be sufficient tomaintain significant activity was not influenced by the presence of cholesterol. Since thedark effect of 0.05 µg/ml of hypericin was approximately 50 % plaque reduction, it is likelythat at an even lower concentration the dark effect would be eliminated. If the light-enhancedeffect is isolated from the dark effect, the influence of a singlet oxygen scavenger may bemore evident. The indication seen of slight reduction in the light activity at thisconcentration is consistent with the model that hypericin has both light-enhanced membrane-directed activity and significant activity in the dark. The singlet oxygen-mediated photo-activity appears to be a more important component in the action of the derivative EGK-149,even though it has also shown some activity in the dark .Reduction of activity in the presence of cholesterol indicates but does not prove thenecessity of singlet oxygen in the reaction. More elaborate techniques can be used toascertain the role of singlet oxygen. Cholesterol as a scavenger can produce a number ofoxidation products from free-radical or peroxide reactions (Halliwell and Gutteridge, 1989),but it produces primarily the 5 -a-hydroperoxide from reacting with singlet oxygen (Kuligand Smith, 1973). This molecule can be chemically isolated to be sure that this was theindeed the end product of the scavenger reaction. Singlet oxygen can also be detected byelectron spin resonance measurements, or its effects be shown to potentiated when applied indeuterium oxide which prolongs its lifetime compared to an aqueous medium. It isinteresting that hypericin and its derivative have shown a difference in sensitivity tomodulation of photo-activity by a singlet oxygen scavenger. It seems more likely, however,that this difference is due to the contributing effects of components of the activities that were132independent of light and not due to differences in the singlet oxygen mechanism of action.Therefore, further efforts to confirm the involvement of singlet oxygen were not undertaken.The photodynamic oxygen-mediated antiviral effect is considered to target themembrane system. As the Sindbis virus membrane is derived from the host cell membrane,the virion envelope and the cell membrane that the virus has to penetrate would presentsimilar sites of action. However, a non-cytotoxic concentration of the compounds wascapable of inactivating the virus or inhibiting the infection process. It is possible that thevirus is more sensitive due to the difference in magnitude of size betweeen a cell and a virusparticle, especially since SINV is only -40 nm in diameter. It is also possible that morespecific interactions involving membrane components are involved, and the viral membraneis not identical to the plasma membrane. A major difference is in the protein composition ofthe membranes as the protein components of the viral envelope are entirely virus-coded. Inthe examination of viral proteins for specific effects on membrane components by thecompounds, there appeared to be no direct alteration of the coat proteins that span the lipidbilayer of the envelope. Neither was a cross-linking of proteins such as that reported as beingcaused by hypericin on DHBV or by a-terthienyl on bacterial proteins observed. The proteinbanding showed a retarding in the electrophoretic mobility of the 30 kD capsid core proteinafter exposure to EGK-149 in light. Moraleda et al. (1993) observed some aggregation effecton the DHBV core protein from treatment with hypericin in the dark. The change in thecapsid protein probably contributed to the overall photodynamic activity of EGK-149, eventhough the capsid is surrounded by the membrane envelope. The membrane integrity waslikely to have been disrupted through a characteristic photosensitizer action on the lipidcomponents such as peroxidation of fatty acids. In the time of treatment assays, EGK-149did not show a post-infection effect, which would potentially result from defective capsidproteins interrupting the progeny virus production. The change in the protein was found intreatment of large numbers of virus particles (5 x 10 5 pfu/ml) with 10 and 100 tg/ml of the133compound. The antiviral assays were conducted using 0.5 and 1.0 1..tg/m1 of compoundagainst virus at 103 pfu/ml, and a concentration as high as 10 .tg/m1 would be cytotoxic inlight. The specific protein-altering photo-active effect of EGK-149 is however intriguingbecause it was not observed with the other photosensitizers hypericin and a—terthienyl. Itshowed that there are different molecular events involved in what is generically described asa photosensitizer effect, even with closely related molecules. The complex sulfur-containingsubstitution groups on EGK-149 may account for this difference in effect with hypericin.The activity of a novel derivative of hypericin brings new information to beconsidered in the saga of deciphering the complex mechanisms of action of hypericin. Theidentification of a light-dependent action on a specific protein is also interesting in terms ofthe molecular mechanism of photodynamic reactions. Studies on the nature of the specificitycan be pursued as the composition of the capsid protein is already known. Investigationshave shown diverse bioactive effects produced by the plant compound hypericin and effortsto clarify the mechanisms of action continue because of its antiretroviral therapeuticpotential. It is a good example of how the elucidation of bioactive natural products can leadto potential therapeutically useful compounds as well as generate research toward theunderstanding of molecular actions134Chapter V. ReferencesAndrei, G.M., Damonte, E.B., de Tone, R.A. and Coto, C.E. (1988) Induction of a refractorystate to viral infection in mammalian cells by a plant inhibitor isolated from leaves ofMelia azedarach L.. Antiviral Res. 9 (4), 221-231.Bonatti, S., Cancedda, R. and Blobel, G. (1979) Membrane biogenesis: in vitro cleavage,core glycosylation, and integration into microsomal membranes of Sindbis virusglycoproteins.. J. 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Virol. 73, 263-272.138Summary DiscussionThe scope of this project included the search and isolation of a biologically activecompound and investigations of the nature of action of another bioactive plant compound. Ina brief summary, this work consisted of the following stages:1. Systematic search of ethnopharmacological information to select medicinalplants for antiviral screening.Documentations of the traditional medicines in Yunnan province of Chinawere screened for plants that have been used to treat diseases that are nowknown to have viral causes.2. Collection and identification of plant specimens.Medicinal plants were collected from three areas of Yunnan to test forantiviral activity.3. Bioassay for antiviral activity.In assays of 31 plant species against two membrane-enveloped viruses,16 showed activity. The most active species selected for isolation of activecomponent(s) was Elsholtzia ciliata of the mint family. This and two otherspecies showed activity enhanced with exposure to long wavelengthultraviolet radiation.4. Purification of active component(s) from active species throughbioactivity-guided fractionation.One active compound isolated from E. ciliata was identified as the polycyclicaromatic hydrocarbon fluoranthene. The presence in the plant of thisanthropogenic compound was probably from environmental accumulation.Other active components of a more polar and possibly ionic nature remain tobe identified.1395. Investigation of nature of activity.Investigation of the mechanisms of action was carried out with thephotosensitive antiviral compound hypericin from medicinal plant of thegenus Hypericum. In structure-activity relationship experiments testingderivatives and relatives of hypericin, a new derivative, 2,5,9,12-tetra(carboxyethylthiomethyl) hypericin (EGK-149), showed potentphotosensitive virucidal activity against enveloped viruses. The activity ofEGK-149 was shown to be of the singlet oxygen type that could be reduced bythe presence of cholesterol as a singlet oxygen scavenger. It also showed theability to alter the Sindbis virus capsid protein with exposure to light whichwas not shown by hypericin.The study covered the different aspects of research in the elucidation of a bioactivecompound even though there was a change from the original plan to find, isolate andinvestigate the mechanism of action of one type of compound when the latter studies weredone with the known plant compound hypericin. As it turned out, the hypericin compoundsprobably presented a better system of study as the compound fluoranthene purified fromElsholtzia is probably not a plant constituent. Its isolation from a bioactivity screening ofmedicinal plants raised instead quite a different set of questions regarding bothphytochemical methodologies and implications relating to environmental quality. While itwas the aim of the project to be comprehensive of the different aspects of natural productresearch, it was understood that in order to do so the work in each area would have to belimited to a certain extent. Each of the aspects covered have the potenial of being done moreextensively and they have each raised the need for further research.The importance to preserve ethnopharmacological information was demonstrated bythe fact that five of the sixteen active antiviral species were only selected on the basis of their140uses in the traditional medicine of ethnic minority tribes in Yunnan. Plants are obviously richresources of bioactive compounds and have played a significant role in providing usefuldrugs or prototypes of drugs as well as experimental research tools. The progress in theunderstanding of the causes of diseases is leading to the refinement of the bioassays to targetfor curative agents. New in vitro assay systems such as ones based on measuring receptorbinding or enzyme activity allow for the relatively rapid screening with small amounts ofplant material for specific activities. Plants remain a wealth of potential for new bioactivityscreening approaches but the threatened loss of biodiversity from the massive destruction ofecosytems like the tropical rainforest imparts urgency to promote conservation.Knowledge about viruses is in the process of rapid developement. It is likely thatviral assay systems are also undergoing developments and more standardization that will leadto more effective antiviral screening programs allowing for testing against a larger battery ofvirus types or more specific disease viruses. It would interesting to examine the efficacy ofplants used in traditional medicine by testing them against the causative agents of thediseases they were used to treat. However, the protocols of testing against specific humandisease viruses was beyond the scope of this project.The results on the chemistry of plants of the genus Elsholtzia have raised somequestions to be answered. As there is another UV-active fraction in E. ciliata, it remains tobe determined if the plant contains any photo-active compounds of plant origin. This isinteresting because photosensitizers have not been reported from the family Lamiaceae. Thepossibility that some plants have a scavenging effect of lipophilic environmental compoundsis worthy of consideration because of the roles many plants have as food and medicine and asa major component of the global environment.141The mechanisms of action of hypericin are not fully elucidated. Similarities anddifferences between the activities of hypericin and the novel derivative EGK-149 add moredata to be considered in the process of resolving the mechanisms. The work to distinguishthe mechanism of action of a compound is complicated as demonstrated by the fact that fewhave been precisely identified to the molecular level. It was probably advantageous to workwith a compound like hypericin for which there is a foundation of existing information on thenature of activity. The quantity and variations of the reported information, however,illustrate again the complexity of the task.It was my goal to conduct a project that combined the different disciplines involved inthe process of identifying a biologically active molecule and understanding how it works.These disciplines of pharmacognosy are: ethnobotany, microbiology, chemistry andpharmacology. I believe that the five aspects of this project have accomplished this goal andthat in addition to my gain in knowledge in each of the fields involved, the integratedperspective of the whole endeavor was an important education.142Appendix IMedicinal Plants Assayed for Antiviral Activityand Their Ethnopharmacological Indications for TreatmentPlant Family^Species^ Part Used #Apiaceae^Centella asiatica (L.) Urban *^ PLcold, infectious hepatitisApocynaceae^Plumeria rubra var. acutifolia RT(Poir.) Ball.hepatitisAsteraceae^Bidens pilosa L. *^ PLcold, prevent flu, hepatitis, jaundiceBerberidaceae^Mahonia nepalensis DC. PLhepatitisCombretaceae^Quisqualis indica L. *^ PLhepatitisConvolvulaceae^Dichondra repens Forst. * PLflu, hepatitis, jaundiceCycadaceae^Cycas siamensis Miq. *^ LFhepatitis, hepatitis A, jaundiceEbenaceae^Diospyros kaki L.f. BKhepatitis, jaundiceEuphorbiaceae^Euphorbia prolifera Ehrenb. ex. Boiss. *^PLhepatitisRicinus communis L.^ RThepatitis, jaundiceFabaceae^Kummerowia striata (Thunb.) Schindl.^PLcold, hepatitis AGentianaceae^Halenia elliptica D. Don^ PLhepatitisIridaceae^Belamcanda chinensis DC. * BUflu, hepatitis143144Plant Family^Species^ Part Used #Lamiaceae^Acrocephalus indicus Briq.*^ PLcoldElsholtzia densa Briq. * PLfluElsholtzia ciliata (Thunb.) Hylander *^BRcold, fluPerilla frutescens (L.) Britton^ PLcold, fluRabdosia phyllostachys (Diels) Hara *^PLcold, hepatitis, jaundiceScutellaria orthocalyx Hand.-Mazz.^PLcoldStachys kouyangensis (Vaniot) Dunn *^PLhepatitis A and BMyrsinaceae^Embelia sessiliflora Kurz *^ RThepatitisOxalidaceae^Oxalis corniculata L. PLcold, hepatitis, jaundicePoaceae^Phyllostachys sp. (gold bamboo)^ PLhepatitisPolypodiaceae^Stenoloma chusanum (L.) Ching PLcold, flu, infectious hepatitisRubiaceae^Hedyotis uncinella Hook. et Am.^PLprevent and cure jaundice hepatitisRutaceae^Boenninghausenia sessilicarpa Levl. *^PLcold, hepatitisSauruaceae^Houttuynia cordata Thunb.^ PLcold, flu, jaundice hepatitisScrophulariaceae^Siphonostegia chinensis Benth. * PLhepatitis A and BVerbenaceae^Verbena officinalis L. *^ PLinfectious hepatitis# Part used: BK, bark; BR, branch; BU, bulb; LF, leaf; PL, whole plant; RT, root.* Showed antiviral activity in bioassay.Appendix II.Literature on the Chemistry of ElsholtziaOn essential oilIn English:Composition of Elsholtzia polystachya leaf essential oil (1988)Ahmed, A., Siddiqui, M.S. and Missra, L.N., Phytochem. 27 (4), 1065-7.Terpenoids from Elsholtzia species. III. New constituents of essentia oil from Elsholtziapilosa (1988)Bestmann et al., Z. Naturforsch. C: Biosci. 43 (5-6), 370-372.Terpenoids from Elsholtzia species. II. Constituents of essential oil froma new chemotype ofElsholtzia cristata (1987)Kobold et al., Planta Medica 53 (3), 268-71.* Constituents of essential oil of Elsholtzia strobilifera (1985)Bisht, J.C., Pant, A.K., Mathela,^Kobold, U. and Vostrowsky, O.,Planta Medica 50 (5), 412-414.Volatile constituents from Elsholtzia polystachya (1984)Nigam, S.S., Saxena, V.K. and Chaturvedi, S.K., Indian Perfum. 28 (2), 71-73.* Gas chromatographic examination of the essential oil of Elsholtzia blanda (1983)Patra et al., Parfuem. Kosmet. 64 (12), 688-92.Composition of essential oil of Elsholtzia strobilifera Benth. (1980)Murari, N.D. and Mathela, C.S., J. Indian Chem. Soc. 57 (10), 1033-4.* Essential oil of Elsholtzia pilosa (1976)Thappa et al., Indian J. Chem. sect. B, 14B (5), 387-388.Chemical studies on the essential oil of Elsholtzia densa (1971)Vashist, V.N. and Atal, C.K., Flavour Ind. 2 (1), 47-48.* Caryophyllene epoxide from the oil of Artemesia scoparia, Elsholtzia polystachya, Piperhookeri, and Piper brachystachyum.(1970)Thappa et al., Curr. Sci. 39 (8), 182-183.Essential of Elsholtzia polystachya (1967)Vashist et al., Indian J. Chem. 5 (3), 130.Elsholzidiol and Elsholtzione (chemistry data)In: CRC Handbook of Terpenoids, Monoterpenoids: Vol. I. pp 126-127.In Chinese:A preliminary study of the acclimatization and essential oil components of Elsholtzia blanda(1989)Cheng et al., Yunnan Zhiwu Yanjiu 11 (1), 91-6.145* Composition of the essential oil of Elsholtzia blanda (1989)Zhu, G.P. and Zhou, W.S., J. Beijing Coll. Trad. Chin. Med. 12 (4), 40-41.Components of essential oils of Elsholtzia stautonii Benth. (1989)Du, H.Q., Zhao, X. and Fang, H.J., Yaowu Fenxi Zazhi 9 (1), 18-21.* Studies on the components of essential oils of Elsholtzia splendens and Origanum vulgare(1983)Li, Z.W. and Zhou, T.H., Yaoxue Xuebao 18 (5), 363-368.In Russian:Characterization of essential oils of different Elsholtzia ciliata biotypes (1988)Bakova et al., Izv. Timiryazevsk. S-kh. Akad. (6), 162-166.Composition of Elsholtzia stautoni essential oil (1987)Dmitriev et al., Izv. Timiryazevsk. S-kh. Akad. (5), 167-170.* Accumulation of essential oils in Elsholtzia patrinii and Lophanthus anisatus (1987)Simonov, I.N., Pavlova, T.A. and Demvanov, P.I. , Izv. Timiryazevsk. S-kh. Akad.(5), 195-199.* Essential oil of Elsholtzia patrinii (1984)Dmitriev et al., Izv. Timiryazevsk. S-kh. Akad. (3), 171-175.Study of some biological characteristics of new essential oil producing plants, Elsholtziapatrinii and Lophanthus anisatus in the Crimea (1980)Barannikova, T.A., Dokl. TAKha, 266, 56-61.In Polish:Composition of Elsholtzia patrinii oil (1969)Czuba, W. and Wozniak, M., Czas. Tech. (Krakow), M. (5), 32-33.In Japanese:Essential oil of Elsholtzia ciliata (1967)Fujita, Y., Tanaka, Y. and Iwamura, J., Nippon Kagaku Zashi, 88 (7), 763-6.In German:ElsholtziaIn: Chemotaxonomie der Pflanzen Vol. IV (1966),Hegnauer, R., Birkhauser Verlag, Basel, pp. 311-312.On other chemical componentsSix flavonoids in Elsholtzia densa Benth. (1991)Zheng, S., Yin, Z. and Shen, X., Bull. of the Northwest Normal Univ., Lanzhou , 27,33-36.146Flavonoids of Elsholtzia cristata (1988)Lee et al., Arch. Pharmacol. Res. 11 (3), 247-249.* Flavonoids of Elsholtzia ciliata (1985)Park, J.H., Woo, W.S. and Shin, K.H., Korean J. Pharmacog. 16 (1), 43- A.* Phytochemical screening of Korean medicinal plants. (III) (1981)Chi, H.J. and Lee, S.Y., Ann. Rep. Nat. Prod. Res. Inst. Seoul Nat. Univ. 20, 38-41.* Screening of saponins in the plants (1981)Han, B.H., Lee, E.B. and Woo, W.S., Ann. Rep. Nat. Prod. Res. Inst. Seoul Nat.Univ. 20, 49-54.A chromatographic survey of anthocyanins in the flora of Japan. I. (1980)Yoshitama, K., Ishii, K. and Yasuda, H., J. Fac. Sci. Shinshu Univ. 15 (1), 19-26.* Screening of Indian plants for biological activity. Part VIII. (1978)Atal, et al., Indian J. Esp. Biol. 16, 330-349.* Occurrence of alkaloids in Korean medicinal plants (1978)Woo et al., Soul Taehakkyo saengyak yonguso opjukjip 17, 17-19.The occurrence of iridoid glycosides in the Labiatae (1972)Kooiman, P., Acta Bot. Neerl. 21, 417-427.Structure of elsholtidiol, a new bisubstituted furan of Elsholtzia densa (1970)Vashist, V.N. and Atal, C.K., Experientia, 26 (8), 817-818.Phytochemical investigationof Elsholtzia cristata (1966)Puziene, G., Liet. TSR Aukst. Mokyklu Mokslo Darb. Med. 9, 147-157. (in Russian)Pharmacobotanical analysis of species of the genus Elsholtzia. II. (1964)Swieboda, M., Diss. Pharm. 16 (1), 121-128. (in Polish)On chemical synthesisA new and short synthesis of dehydroelsholtzione (Naginata ketone) and isoegomaketone(1980)Pilot et al., Tetrahedron Lea. 21 (49), 4717-20.New synthesis of elsholtzia ketone (1977)Cazes, B. and Julia, S., Synth. Commun. 7 (1), 113-117.* = cited and % of compounds present calculated in NAPRALERT database, issue 2, 1992.(Program for Collaborative Research in the Pharmaceutical Sciences, Dept. of MedicinalChemistry and Pharmacognosy, College of Pharmacy at the University of Illinois,Chicago).(This information is compiled from the following sources: the NAPRALERT database,computer literature searches through the UBC library system and literature seen.)147cm-1Appendix IVHigh Resolution Mass Spectrum Fragment Analysis of Purified Fluoranthene(By UBC Department of Chemistry Mass Spectroscopy Centre DS-55 MS data sytem)DPZ:NDATA:H10952.MSSCAN:^2,^1/27/93^16:^SIONISATION:^ElNO.^PEAKS:^296BASE/NREF^INT:^107072./^62623.TIC:^823568.MASS^RANGE:^30.9984^-^680.9569RETN TIME/MISC: 7:27/1051/^2/ 100PAGE 1 PAGE 2C16H N204DEV MEAS MASS #PTS^XINT C^H16N204DEV MEAS MASS #PTS XINTNONE 294.9962 4^0.29 10^11 2 2 1.715 1 2 4 3.5 272.9971 4^0.37 15^10 0 0 1.2 190.0795 4 0.2714 2 1 4 -4.7 247.9937 6^0.52 NONE 181.9827 4 0.3013 0 2 4 -0.9 247.9849 4^0.45 11^3 1 2 -0.6 181.0157 4 0.30NONE 240.9844 8^2.52 14^10 0 0 -2.5 178.0758 12 0.77NONE 224.9844 5^0.339^10 2 2 1.514 2 0 3 0.9 218.7212 6^0.3310^10 1 2 -1.8 176.0694 8 0.81NONE 217.9935 6^0.3214^89^80202-1.42.6176.0612 10 1.1612 1 0 4 2.0 208.9394 4^0.20 14^7 0 0 -0.8 175.0540 17 1.4512 1 1 3 1.2 206.9368 5^0.379^7 2 2 3.316 12 0 0 -4.8 204.0891 12^0.9113^5 1 0 3.2 175.0454 4 0.2311 12 2 2 -0.8 ....40■14^6 0 0 0.2. 174.0467 2S 4.6215 10 1 0 -0.4 204.0809 4^0.239^6 2 2 3.812 12 0 3 2.3 14^5 0 0 0.8 173.0399 4 0.2916 11 0 0 -4.1 203.0820 29^16.919^5 2 2 4.811 11 2 2 -0.1 13^0 1 0 -3.4 169.9996 5 0.2712 14 2 1 -4.8 202.1758 4^0.20 10^2 0 3 -0.79 16 1 4 -2.110^3 1 2 -2.3 169.0140 5 0.27m40.1611101002020.44.4'202.0786 51^100.0214^19^10202-1.42.6169.0064 5 0.291611990202-0.23.8201.0702 29^15.43 13^78^70202-2.91.1163.0519 4 0.4616118802020.94.9200.0635 35^18.5013^08^002020.64.6156.0006 4 0.2211 4 0 4 -2.9 220.0.082 4^0.2112^8 0 0 -1.7 152.0609 17 1.3316 7 0 0 0.7 199.2554 21^3.607^8 2 2 2.311 7 2 2 4.712^7 0 0 0.6 151.0554 8 0.3816 6 0 0 2.5 198.2495 12^1.977^7 2 2 4.716 5 0 0 -3.2 197.2359 6^0.317^7 2 2 -1.7 151.0491 4 0.2611 5 2 2 0.8 10^1 1 1 2.1 151.0079 4 0.2315 12 .2 0 0.7 192.2346 12^1.217^3 0 4 4.810 12 2 2 4.7NONE 150.9855 4 0.2215 11 0 0 -2.4 191.2837 6^0.5412^6 0 0 3.2 150.0502 5 0.78149PAGE 3 PAGE 4C H N 0 DEV MEAS MASS #PTS %INT C H N 0 DEV MEAS MASS #PTS %INT16 2 4 16 2 43 3 2 2 3.612 6 0 0 -2.4 150..0445 10 0.967 6 2 2 1.6 3 3 2 2 - 3.5 99.0159 5 0.3112 5 0 0 0.4 149.0295 5 0.37 8 2 0 3.6 92.0192 8 0.457 5 2 2 1.4 4 4 1 -4.98 3 1 2 -2.4 145.0140 6 0.42 8 2 0 .0 -1.0 98.0145 12 1.183 2 2 2 3.010 1 0 1 -3.6 136.9992 8 0.635 1 2 3 0.5 7 12 0 0 -13.9 96.0930 6 0.3210 0 0 1 1.4 125.9962 6 0.41 7 11 Z 0 1.3 05.0874 5 0.4711 2 0 0 -1.0 134.0145 4 0.22 7 5 Z Z 2.1 89.0'412 4 0.246 2 2 2 3.0 ■IPw-7 4 0 0 0.2 88.0315 29 8.788 3 0 2 -1.7 131.0116 5 0.26 2 4 2 2 4.33 3 2 4 2.37 3 0 0 1.3 87.0248 14 2.8010 7 0 0 1.0 127.2557 5 0.415 7 2 2 5_0 7 3 0 0 -3.3 87.0202 8 0.912 3 2 2 0.710 6 0 0 -0.8 126.0461 4 0.395 6 2 2 3.2 7 2 0 0 2.6 86.0163 4 0.372 2 2 2 4.79 1 2 1 -3.8 124.9992 12 2.824 1 2 3 0.3 NONE 85.3016 4 5.127 3 0 2 -3.4 119.0099 8 0.47 NONE 84.9692 5 0.722 3 2 4 0.6 NONE 83.9558 10 0.719 3 0 0 0.6 111.0241 6 2.394 3 2 2 4.7 NONE 83.9518 6 0.409 2 2 0 -3.2 110.0124 4 0.22 6 11 0 0 0.4 83.0865 5 0.304 2 2 2 0.8 4 2 0 2 0.5 82.2260 8 2.578 13  0 2 -1.8 109.0999 5 0.28 6 9 0 0 -1.2 81.0692 8 0.44NONE 101.5408 14 2.284 1 0 2 1.3 80.9989 4 0.394 7 1 2 -1.1 101.0/66 5 0.38 6 4 0 0 2.5 76.0338 6 0.368 5 0 0 -2.4 101.0387 25 13.523 5 2 2 3.6 6 3 0 g 1.4 75.0248 10 1.53NONE 1130.5371 21 2.36 6 3 0 0 -2.2 75.0213 4 0.291 3 2 2 1.8.40. 8 4 0 2 2.3 120.0316 35 12.243 4 2 2 4.3 6 2 0 2 1.5 74_2171 21 1.37NONE 99.5271 12 1.24 NONE 69.9985 21 2.938 3 0 0 -0.3 99..0732 21 3.19 5 9 0 2 -2.3 69.2721 6 2.37150

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