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Thiarubrine production in roots and root cultures of Ambrosia chamissonis Ellis, Shona Margaret 1993

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THIARUBRINE PRODUCTION IN ROOTS AND ROOT CULTURESOF AMBROSIA CHAMISSONISbySHONA MARGARET ELLISB.Sc., The University of British Columbia, 1985A THESIS SUBMII I ED IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIES(Department of Botany)We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJULY 1993© Shona Margaret EllisIn 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 eBOTA NJ The University of British ColumbiaVancouver, CanadaDate TueIZO ci cl 3DE-6 (2/88)ABSTRACTAmbrosia chamissonis (Asteraceae) provides antibiotic sulphur heterocyclic polyynesknown as thiarubrines and thiophenes. They occur in all parts of the plant, especially in the rootsand can be visualized in hand sections. Methods were developed for their isolation, purificationand quantification. Plants were collected from various locations in B. C. and found to havedifferent thiarubrine profiles. A new thiarubrine (3-(1-propyny1)-6-(6-hydroxyhex-3-yn-lyny1)-1,2-dithiacyclohexadiene) and thiophene (2-(1-propyny1)-6-(6-hydroxyhex-3-yn-1yny1)-thiophene) were identified from roots and root cultures. Transgenic and non-transgenic rootcultures were generated and shown to produce the same compounds as occur in natural roots.Quantitative chemical profiles of the cultured roots matched those of roots which had littlesecondary growth. Elicitation, with fungal preparations, did not enhance the over-all thiarubrineconcentration, but the content of a more fungicidal thiarubrine was enhanced at the expense of aless active, but predominant thiarubrine. Administration of biosynthetic precursors did not haveany effect on thiarubrine production. Antibiotic testing indicated that the thiarubrines are toxicagainst a number of fungi and, to a lesser degree, bacteria.TABLE OF CONTENTSABSTRACTLIST OF TABLESLIST OF FIGURES^ viACKNOWLEDGEMENTS ixCHAPTER ONE: GENERAL INTRODUCTION^ 1CHAPTER TWO: ROOT CHEMISTRY OF AMBROSIA CHAMISSONISIntroduction^ 10Materials and Methods^ 12A. Establishment of Extraction, Isolation and PurificationProcedures for Thiarubrines and Thiophenes^ 12B. Extration in Spring and Autumn of Roots from TwoCollecting Sites^ 15Results^ 16Discussion 30CHAPTER THREE: COMPARISON OF THIARUBRINE AND THIOPHENEPRODUCIION BETWEEN ROOTS AND ABOVE GROUND PORTIONS OFAMBROSIA CHAMISSONISIntroduction^ 32Materials and Methods^ 33Results^ 35Discussion 40CHAPTER FOUR: IDENTIFICATION OF A NOVEL THIARUBRINE ANDTHIOPHENEIntroduction^ 42Materials and Methods^ 43ivResults^ 44Discussion 50CHAPTER FIVE: ESTABLISHMENT AND ANALYSIS OF ROOT CULTURESIntroduction^ 52Materials and Methods^ 56Results^ 61Discussion 71CHAPTER SIX: ELICITATION WITH FUNGAL CELL WALL PREPARATIONSIN HAIRY ROOT CULTURES OF AMBRIOSIA CHAMISSONISIntroduction^ 94Materials and Methods^ 77Results^ 79Discussion 82CHAPTER SEVEN: ANTIBIOTIC AND ANTIFUNGAL ACTIVITY OF THETHIARUBRINESIntroduction^ 84Materials and Methods^ 87Results^ 91Discussion 97CHAPTER EIGHT: GENERAL SUMMARY^ 101REFERENCES^ 103APPENDICES 109LIST OF TABLESTable 1.1: Distribution of Thiarubrines.Table 2.1: Rf Values of the Thiophenes on Preparative Thin LayerChromatography on Silica Gel F254.Table 2.2: Analytical HPLC Retention Times (in Minutes) of the Thiarubrinesand Thiophenes with Different Solvent Systems (H20:CH3CN).^22Table 2.3: Preparative HPLC Retention Times (in Minutes) for the Purificationof Thianibrines and Thiophenes with Different Solvent Systems.^23Table 3.1: Overall Thiarubrine Content of the Different Organs of Ambrosiachwnissonis.^ 36Table 5.1: Overall Thiarubrine Content of the Root Cultures.^ 63Table 5.2: Thiarubrine Yield From Freeze-Dried Cultures from Fermenter.^67Table 5.3: Thiarubrine Production in Hairy Root Cultures (A4) Treated withMagnesium Sulphate.^ 68Table 5.4: Thiarubrine Production in Transgenic Cultures Treated with VariousConcentrations of Malic Acid. 69Table 6.1: Overall Thiarubrine Production of the Hairy Root Cultures Elicitedwith Ping Elicitor.^ 79Table 7.1: Disk-Diffusion Assay to Determine the Relative 'Foxicities ofThiarubrines to Micro-Organisms in Dark and 2 hr Exposure to UV-AExpressed as Zones of Growth Inhibition (mm).^ 92Table 7.2: Disk-Diffusion Assay to Determine Antimicrobial Activity ofThianibrines Against C. albicans on Different Media in Dark and2 hr Exposure to UV-A, Expressed as Zones of Growth Inhibition (mm).^93Table 7.3: Disk-Diffusion Assay to Determine the Dark Toxicities of Thiarubrinesto Fungi Expressed as Zones of Growth Inhibition (mm).^93Table 7.4: Comparison of the LC50 and Minimum Inhibitory Concentrations(MIC) For P815 and Canthda albicans.^ 95Table 7.5: Toxicity Indices of thiarubrines for P815 and C. albicans.^95Table 7.6: Broth Dilution Assay to Determine Minimal Inhibitory Concentration(MIC) for (A) P. aeruginosa in the Dark vs. 2 hr Exposure to UV-ALight and (B) A. fumigatus fortwo Different Media.^ 96420LIST OF FIGURESFigure 1.1: Thiarubrines and Thiophenes Characteristic of Ambrosia chamissonis.^2Figure 1.2: Sulphur Extrusion from Thiarubrine 1 a after Irradiation withUV-A Light^ 5Figure 1.3: Biological Activity of Thiarubrine la.^ 7Figure 2.1: Collection Sites in British Columbia, of Ambrosia chamissonis.^11Figure 2.2: Extraction Protocol for Thiarubrines and Thiophenes fromRoots of Ambrosia chamissonis.^ 13Figure 2.3: Localization of Thiarubrine Canals in Woody Roots ofAmbrosia chamissonis (Cross-Section). 16Figure 2.4: Localization of Thiarubrine Canals in Non -Woody (Succulent)Roots of Ambrosia chamissonis.^ 17Figure 2.5: HPLC Trace at a Flow Rate of lml/min at (i) 28:72 (H20:CH3CN)and (ii) 50:50 (H20:CH3CN). 18Figure 2.6: HPLC Trace of Crude Root Extract at 340 nm and 490 nm at aFlow Rate of 1 ml/min with H20:CH3CN (28:72).^ 19Figurre 2.7: Mass Chromatogram and Spectrum of a Mixture of Thiarubrines1 a and 2 a Isolated by Column Chromatography. 24Figure 2.8: (i) GC-Mass Chromatogram of the TMS-Derivative of Thiophenes4b, 5 b, 6b, 7b, and 8b. (ii) Mass Spectrum of the TMS-Derivative ofThiophene 7 b.^ 25Figure 2.9: (i) GC-Mass Spectrum of the TMS-Derivative of Thiophene 4band (ii) Solid Probe Mass Spectrum of Thiarubrine 4a Crystallizedfrom Preparative HPLC Fraction.^ 26Figure 2.10: GC-Mass Spectra of the TMS-Derivatives of ChlorinatedThiophenes 5 b and 6b.^ 27Figure 2.11: HPLC Traces of Roots Collected from Plants with Two Typesof Leaf Morphology from the Queen Charlotte Islands.^ 28Figure 2.12: Thiarubrine Content of Collections from Centennial Beach (CB)and the Queen Charlotte Islands (Q) in the Spring and Autumn^28Figure 2.13: Thiarubrine Profiles of Collections from Centennial Beach (CB)and the Queen Charlotte Islands (Q) in the Spring and Autumn.^29Figure 2.14: Proposed Biosynthetic relationship Between some of theThiarubrines.^ 31Figure 3.1: Thiarubrine Canals in the Young Stem (i) and Leaf (ii) ofviviiAmbrosia chamissonis.^ 35Figure 3.2: Percent of Thiarubrines (1 a, 2 a, 3 a, 4 a, 7 a, and 8 a) andThiophenes (1 b, 2 b, 3 b, 4 b, 7 b, and 8 b) from the Total PolyyneContent of the Root Extracts. 37Figure 3.3: Percent of Thianthrines (l a, 2a, 3 a, 4a, 7 a, and 8 a) andThiophenes (1 b, 2 b, 3 b, 4 b, 7 b, and 8 b) from the Total PolyyneContent of the Woody Stem Extracts.^ 37Figure 3.4: Cross-Section Through the Green Stem with Thiarubrine Canals ofAmbrosia chamissonis.^ 38Figure 3.5: Percent of Thiarubrines (la, 2 a, 3a, 4a, 7a, and 8a) andThiophenes (1 b, 2 b, 3 b, 4 b, 7 b, and 8 b) from the Total PolyyneContent of the Green Stem Extracts.^ 38Figure 3.6: Cross-Section Through the Leaf, with Thiarubrine Canals ofAmbrosia chamissonis.^ 39Figure 3.7: Percent of Thiarubrines (l a, 2a, 3 a, 4a, 7 a, and 8 a) andThiophenes (1 b, 2 b, 3 b, 4 b, 7 b, and 8 b) from the Total PolyyneContent of the Crude Leaf Extracts.^ 39Figure 4.1: "In Flight" UV-Visible Spectra of Thiophene 7 b and 8 b from HPLC.^45Figure.4.2: UV-Visible Spectrum of Thiarubrine 8 a.^ 46Figure 4.3: High Resolution Mass Spectrum of Thianibrine 8a.^ 46Figure 4.4: GC-Mass Chromatogram of Sample Isolated by TLC. 47Figure 4.5: GC Mass Spectrum of Thiophene 8 b and Fragmentation Pattern ofthe Molecule.^ 48Figure 4.6: GC Mass Chromatogram of the TMS Derivatives of theHydroxylated Thiophenes.^ 49Figure 4.7: 300 mHz 1H NMR of Thiophene 8b.^ 50Figure 5.1: Transformation of Ambrosia chamissonis withAgrobacterium rhizogenes.^ 53Figure 5.2: Transformation of Ambrosia chamissonis stem Segments withAgrobacterium rhizogenes. 57Figure 5.3: Comparison of Thiarubrine Composition in Roots and RootCultures.^ 63Figure 5.4 Cross-Section of a Transgenic Root. The Thiarubrine Canals areEvident between the Double Endodermis.^ 64Figure 5.5: Cross-Section of a Transgenic Root. The Thiarubrine Canals areEvident between the double Endodermis.^ 64Figure 5.6: Growth Curves of Transgenic Root Cultures and Normal RootCultures.^ 65Figure 5.7: Growth Curve of Transgenic Root Cultures and ThiarubrineProduction (in mg/ g dry wt.).^ 65Figure 5.8: HPLC Trace of an 8 Day Old Root Culture Extract at a Flow Rateof 1 ml/min at 50:50 (H20:CH3CN). 66Figure 5.9: Thiarubrine Content of the Cultured Roots (HR=Hairy rootstransformed with Agrobacterhan rhiwgenes A4, NR= Normal Rootcultures) and the Culture Medium.^ 67Figure 5.10: HPLC Traces of Untreated Versus Treated Cultures with Malic Acidand UV-Vis Spectrum of Thiosulphinate 9.^ 69Figure 5.11: HPLC Trace of Extract from Malic Acid Experiment RepresentingTreated and Untreated Cultures with the "In Flight" UV-Vis Spectra ofUnknown Components.^ 70Figure 6.1: The Effect on Thiarubrine Profile of Hairy Root Cultures Elicitedwith Varying Concentrations of Pmg Elicitor-Trial 1.^ 81Figure 6.2: The Effect on Thiarubrine Profile of Hairy Root Cultures Elicitedwith Varying Concentrations of Pmg Elicitor-Trial 2. 81viiiACKNOWLEDGEMENTSThere are many people I would like to express my gratitude to for their help and support. Iwould especially like to thank Felipe Balza for his guidance with the chemical analyses, ZytaAbramowski for her help with the antibiotic work, my supervisor Neil Towers for his input andencouragement, and Herbert Kronzucker for his translation of German papers. Among my friendsin the Department I would like to express my appreciation to Dr. Jack Maze and Dr. Carl Douglasfor their contributions and direction as members of my committee, Dr. lain Taylor who gave memy first job in the Botany Department, as well as Dion Durnford, Paul Spencer, Lynn Yip, CarolAnn Borden, Rosemary Mason, Terry Jarvis, Phil Gunning and the Botany Office staff for theirfriendship and support. I would also like to thank my family for their patience and encouragementthroughout my studies.I wish to give a very special acknowledgement to Dr. Wilf Schofield for his contagiousenthusiasm for botany which directed me as an undergraduate.ixCHAPTER ONEGENERAL INTRODUCTIONAmbrosia chamissonis grows on sandy maritime beaches along the west coasts of Northand South America. Its range extends from Baja California to the Queen Charlotte Islands ofBritish Columbia in North America. In South America it was first reported in Chile in 1892 on Islade la Mocha (Nakatani et al., 1973). It has spread northward and occupies several hundred milesof coastal Chile. There have been problems with the taxonomy of this species due tomorphological variability. Payne (1964) re-evaluated Ambrosia and combined a number of speciesof Ambrosia with Franseria to form the heteromorphic species A. chamissonis. The taxonomy isbased on the nature of the spines of the fruiting involucres rather than on leaf morphology. Leafmorphology varies within and between geographic regions. The group of chemicals mostextensively studied in this group are the sesquiterpene lactones, but the analyses of thesecompounds have supplied little chemotaxonomic information (Payne et al., 1973). Like leafmorphology the chemical production of the plant is variable. However, Nakatani eta!. (1973)point out that the Chilean variety is relatively uniform in both chemistry and morphology. Theysuggest that this may be due to the introduction of the species and subsequent spread to other sitesmaking this a genetically uniform line. The chemistry and morphology mimic that of the plantsfound north of San Francisco implying that this may be the origin of the Chilean strain. The onlyother relatively uniform population is found in southern California and is cut off physically fromthe rest of the North American population (by extensive cliffs). The genus Ambrosia, isconsidered a highly evolved member of the Family Asteraceae in the tribe Heliantheae and subtribeAmbrosiinae. Bohlmatui (1990) points out that the subtribes can be separated based on their mainchemical constituents. Information on acetylenes and sesquiterpenes are both useful.1R2S—SaThiarubrines: la, 2a, 3a,4a, 5a, 6a, 7a, 8a.Thiophenes: lb, 2b, 3b,4b, 5b, 6b, 7b, 8b.R21^ CH22 CH3— (C ="=----^ CC—cH=cH23 CH3—C---C^(C-7=-C)2— CH— CH2— C)2 — CHOH — CH2OH5 CH3 Ca=—=C^(C^— CHCI— CH2OHCHOH— CH2C17 HO — CH2-0-=—=C^CH= CH28 CH3—C---C^q—c H2— CH2OH2Figure 1.1 Thiarubrines and Thiophenes characteristic of Ambrosia chamissonis.Members of the Asteraceae characteristically produce polyynes. These acetyleniccompounds are also found in the Campanulace,ae, Apiaceae, Araliaceae and a number of other plantfamilies as well as in the Basidiomycetes (Bohlmann et aL, 1973). A number of sulphurderivatives including dithiacyclohexadiene polyynes and thiophenes are found exclusively in theAsteraceae primarily in the Heliantheae. The dithiacyclohexadiene polyynes, commonly calledthiarubrines (Fig. 1.1), are found in a restricted number of genera. For example, Chaenactisdouglasii and Eriophyllum lanatum produce the positional isomers thiarubrine 1 a, 3-(1-propyny1)-6-(5-hexen-3-yn-l-yny1)-1,2-dithiacyclohexa-3,5-diene and thiarubrine 2 a, 3-(pent-3-yn-1-yny1)-6-(3-buten-1-yny1)-1,2-dithiacyclohexa-3,5-diene (Norton et. al., 1985a). These have beentrivially called thiarubrines A and B respectively (Norton et. al., 1985a). For simplicity thethiarubrines and thiophenes will be assigned numbers and letters in this paper (Fig. 1.1 andAppendix 1). Thiarubrines are designated with the letter "a" and thiophenes with the letter "b".Rudbeckia hirta produces geometric isomers of thiarubrine 10a (Constabel et al., 1988). Table1.1 summarizes the distribution of the thiarubrines throughout the Asteraceae (the only family theyoccur in) following the taxonomic scheme of Robinson (1981). The thiarubrines are convertedinto their corresponding thiophenes via sulphur extrusion by irradiation (Fig. 1.2). Thiarubrine 1 ayields thiophene l b (2-(1-pmpyny1)-5-(5-hexen-3-yn-1-yny1)-thiophene) and 2a produces 2 b (2-(pent-3-yn-lyny1)-5-(3-buten-l-yny1)-thiophene).Ambrosia chamissonis has been found to produce the widest array of thiarubrines of anyplant analyzed to date(Balza and Towers, 1990, 1993). Thiarubrines 1 a and 2a, are present inlarge quantities. Other thiarubrines, based on the thiarubrine 1 a skeleton, have been identified.Thiarubrine 3 a, 3-(1-pmpyny1)-6-(5,6-epoxyhex-3-yn-1-yny1)-1,2-dithiacyclohexa-3,5-diene, anepoxide and thiarubrine 4 a, 3-(1-propyny1)-6-(5,6-dihydroxyhex-3-yn-1-yny1)-1,2-dithiacyclohexa-3,5-diene, a vicinal diol were found to be unique to this group and in relativelyhigh quantity accompanied by their corresponding thiophenes 3 b, 2-(1-propyny1)-5-(5,6-epoxyhex-3-yn-1-yny1)-thiophene and 4 b, 2-(1-propyny1)-5-(5,6-dihydroxyhex-3-yn-1-yny1)-3Table 1.1 Distribution of Thiarubrines.Species^Thiarubrine^ReferenceTribe EupatorieaeSubtribe AgeratinaeMikania officinalis^ 2*^Bohlmann et aL, 1973Tribe Heliantheae4Subtribe AmbrosiinaeAmbrosia artemisifoliaA. chamissonisA. confertifloraA. cumanensisA. eliatorA. psilostachyaA. trifidaA. trifoliataIva xanthifoliaSubtribe BaeriinaeEriophyllum caespitosumE. lanatumE. staechadifoliumLasthenia chrysostomaL coronariaSubtribe ChaenactidinaeChaenactis douglasiiC. glabriusculaPalafoxia hookerianaP. texanaPicradeniopsis woodhouseiSchkuhria abrotanoidesadvenaS. multifloraS. pinnataS. senecioidesLa^Bohlm,smn and Kleine, 1965la Bohlinann et al, 19772a Norton et al, 1985a3*^Balza et al., 19894a Balza et aL, 19895a Balza and Towers, 19906a^Balza and Towers, 19907a Balza and Towers, 19908a Ellis et al, 19939 Balza and Towers, 1990la^Lopez et al, 1989la Bohlmann et at., 1977La Bohlmann and Kleine, 1965la^Lopez et aL, 1989la Bohlmann and Kleine, 196511a Lu et aL, 1993la BohlmAnn and Kleine, 1965La^Bohlmann and Kleine, 19652 a^Bohlmann and Kleine, 1965la Norton et al, 1985a2a Bohlmann et a/., 1981la^Bohlmann et ai., 198112a Bohlmann and Zdero, 197812a Bohlmann and Zdero, 19781 a^Norton et aL, 1985a2a Norton et al., 1985a2a Bohhnann et ai., 1973La^Bohlniann et aL, 19732a Bohlmann et aL, 197312a Bohlmami et aL, 1976la^Bohlmaiin etaL, 1973la Bohlmann and Kleine, 1965La Bohlmann, Jakupovic, et al, 1980la^Bohlmann and Zdero, 1977Bohlmann and Kleine, 1965la BohLmatin and Kleine, 1965Table Cont.Species^Thiarubrine^Reference5Subtribe EcliptinaeOyedeae boliviana^ 2a^Bohlmann and Zdero, 1979Verbesina alata 2a Bohlmann et aL, 1973V. boliviana 2a Bohlmann, Grenz, et al., 1980V. cinerea^ 2a^Bohlmaim, Grenz, et al., 1980V. latisquamata la Bohlmann and Lonitz,1978aV. occidentalis la Bolilmann and Lonitz, 1978bZexmenia hispida 2a^Bohlmann and Lonitz, 1978cSubtribe MelampodiinaeMelampodium divaricatum^10a (E)^Bohlmann and Le Van, 1977Bohlmann and Kleine, 1965M. longifolium^ 10a (E) Bohlmann and Kleine, 1965Subtribe MineriinaeMilleria quinquefolia^2a^Bohlmann et aL, 1973Subtribe RudbeckiinaeR bicolor^ 10a (E)^Bohlmann and Kleine, 1965R hirta 10a Bohlmann and Kleine, 1965R. newmannii 10a 13ohlmann et aL, 1973R. speciosa^ 10a (E)^Bohlmann and Kleine, 1965R. sullivantii 10a Bohlmann et aL, 1973* The thiarubrines are present with their corresponding thiophenesFigure 1.2 Sulphur Extrusion from Thiarubrine 1a after Irradiation with UV-A Light.thiophene. Compounds present in lower concentrations included two chlorohydrins 5a (341-propyny1)-6-(5-chloro-6-hydroxyhex-3-yn-lyny1)-1,2-dithiacyclohexa-3,5-diene) and 6a (3-(1-propyny1)-6-(6-chloro-5-hydroxyhex-3-yrt-lyny1)-1,2-dithiacyclohexa-3,5-diene), a primaryalcohol 7 a (3-(hydroxyprop-1-yny1)-6-(5-hexen-3-yn-1-yny1)-1,2-dithiacylohex-3,5-diene ) andtheir corresponding thiophenes 5 b, 6 b and 7 b. Thiosulphinate 9 (3-(1-propyny1)-6-(5-hexen-3-yn-l-yny1)-cyclohexa-3,5-diene-1,2-thiosulphinate which exists without a correspondingthiophene.There is little known about the biosynthesis of the 13 carbon thiarubrines and thiophenes,although it is agreed that they originate from acetylenic precursors. The mechanism of triple bondformation in the generation of the acetylene tridecapentaynene as well as the addition of H2S, or itsbiochemical equivalent, to the conjugated triple bonds remain unknown. Tracer studies byBohlmann eta!. (1973) indicated that the terminal carbon of a fatty acid is usually lost in thegeneration of the odd carbon polyynes. This was corroborated by Gomez-Barrios et al. (1992)who used 13C-acetate in their tracer work. Thiantbrine 1 a is believed to be the precursor to theother hydroxylated and chlorinated thiarubrines. Even though the thiophenes can be produced byirradiation of the corresponding thiarubrine, there appears to be a separate biosynthetic pathway inthese plants as demonstrated in Chaenactis douglasii using 35S incorporation (Constabel et aL ,1989b). Bohlmann (1973) proposed that the thiarubrines exist in equilibrium with their thioketoneisomers which could explain the red coloration, however this explanation does not appear likely(Norton eta!. 1985). In Bohlmann's comprehensive book, UV-Visible spectra were presented fora large number of polyynes and much can be gleaned about the structures of these compounds bysuch data.The function of these chemicals is apparently defensive. Thiarubine 1 a and thiophene 1 b,as well as the Rudbeckia thiarubrines and thiophenes, have been tested against the humanpathogens Saccaromyces cerevisiae, Escherichia coli, and Pseudomonas flavescens (Constabel andTowers, 1989c). Light (UV-A) was found to be an important factor in the activity of thethiophenes. The thiarubrines, however, have light-independent as well as a light dependent6Visible LightThiarubrine la^  Thiophene lb(Activated Intermediate)Light-IndependentToxicityVisible Light Dependent UV-A DependentToxicity^Toxicityactivity. The process of conversion from the thiarubrine to the thiophene or the activity of theresulting thiophene may be responsible for the toxicity. P. flavescens was not affected by any ofthe compounds at the concentrations tested. Hudson et al. (1986) examined the activity ofthiarubrines against viruses and found toxicity is light-dependent. The thiophenes are toxic only inthe light at higher concentrations. The thiarubrines are toxic to nematodes and to cockroaches(Towers and Abramowski, unpublished).Figure 1.3 Biological Activity of Thiarubrine la (Constabel and Towers, 1989).Tissue culture has great potential for the study of plant chemistry. To date, however, theutili72tion of plant cell cultures for large scale production of secondary compounds have not beensucessful in many cases. One of the main reasons for this is that unorganised tissues, such ascallus and suspension cultures, which have traditionally been employed, usually do not producethe desired secondary metabolites. On the other hand, organ cultures, such as roots, are goodsources of secondary compounds characteristic of the particular tissue. This principle was nicelydemonstrated by Cosio et al. (1986) who examined crown gall tumour cultures of Chaenactisdouglasii. The transgenic callus produced and accumulated thiarubrines whereas normal callus didnot. Histological inspection of transgenic callus, revealed that they actually resembled roots.Norton eta!. (1985b) examined the production of thiophenes in crown gall tumours and calluscultures of Tagetes patula. The thiophenes were produced at a reduced and inconsistent level. The7tumours apparently had some degree of differentiation into organized tissue where secondarymetabolites were synthesized. Callus cultures derived from tumours produced more thiophenesthan those which were not transgenic, but the levels were low when compared with the plant roots.Differentiated tissue, such as roots, are relatively easy to culture depending on plant species. Rootcultures usually reflect the chemistry of the plant root. Because the thiarubrines are light sensitive,culture conditions as well as extraction procedures must be adapted to accommodate this.One of the drawbacks of tissue culture is that over time the requirements for growth maychange especially with callus and suspension cultures. Root cultures may change their requirementfor growth regulators (ie. auxins and/or cytokinins) over time. "Hairy" root cultures do not requireplant hormones for their growth. Agrobacterium rhizogenes is the causative agent of hairy rootdisease. This plant pathogen transfers a piece of DNA into the plant cell where it is stablyincorporated into the plant nuclear genome. Several genes are involved in this transfer andultimately cause the proliferation of roots at the infection site. Roots can be induced andmaintained in culture.Hairy root cultures are stable for extended periods of time. Aird eta!. (1988) examined thechromosome numbe in hairy root cells from various plant species and found the chromosomenumbers to be identical to that of the parent plants. Both suspension cultures and callus cultures,exhibit chromosomal variation after numerous subcultures. The production of secondarymetabolites in root cultures was shown to be stable over time. There are claims that higherconcentrations of secondary metabolites are produced in transgenic cultures than in untransformedroot cultures (Constabel and Towers, 1989a). Whether these cultures are intrinsically superior orwhether it is the selection of high yielding lines which account for this remains questionable.Manipulations of culture conditions may also enhance the production of importantchemicals, through precursor feeding as well as stimulation of production through the use ofelicitors. Since many desirable compounds are defensive in nature, the exposure to a pathogen or acomponent extracted from a pathogen, may cause the plant tissue to respond by increasing theproduction of defensive chemicals.8The focus of this research was to continue the examination of the thiarubrines andthiophenes of Ambrosia chamissonis as they have medicinal potetnial. Traditionally the roots havebeen examined as the source of these compounds. In these studies, above ground portions werealso investigated. Chemical as well as morphological studies demonstrate the complex nature ofthe production of these compounds. Production in tissue culture, was examined to determine ifcultured plant tissue is an efficient way to produce them. In addition, the activities of the differentthiarubrines against numerous microorganisms were examined.9CHAPTER TWOROOT CHEMISTRY OF AMBROSIA CHAMISSONISINTRODUCTIONThe genus Ambrosia has been re-examined and many species, which have been separatedon the basis of leaf morphology, are now believed to belong to one heteromorphic species (Payneet aL 1964). No correlation was found between leaf morphology and the sesquiterpene lactoneprofiles in plants collected from western North America (Payne et al. 1973). In British Columbiathe Centennial Beach (CB), Tsawassen population is composed of plants with one type of leafmorphology, whereas the North Beach, Queen Charlotte Island (QCI) population is made up ofthree types (Fig. 1.1). The morphologically distinct plants of QCI may grow within a metre ofeach other, the most common type (Type 1) being of similar leaf morphology to the Tsawwassenplants. In the autumn there is more variation within the individuals of the QCI plants. Thechemistry of the roots from two collection sites were examined.The roots of species of Ambrosia have been examined for their acetylene composition(Bohlmann et aL, 1973, Balza et al. 1989, 1990). Thiarubrines (la and 2a) and thiophenes (1 band 2b) were reported as well as pentaynene, a supposed precursor. Balza et al. (1989) haveidentified thiarubrines la and 2a, as well as others which have the basic structure of thiarubrine lain the mots of Ambrosia chamissonis from two populations: Marin County, California andTsawassen, British Columbia. The vicinal diol, thiarubrine 4 a, as well as the epoxide, thiarubrine3a, were identified along with their corresponding thiophenes. Later the structures of othersincluding a primary alcohol (7a), two chlorohydrins (5a and 6a), accompanied with theircorresponding thiophenes, and a thiosulphinate (10) were elucidated (Balza et al., 1990). The lowyield of these compounds made it impossible to quantify them accurately, but it was found that theMarin County Collections had a higher level of the chlorohydrins than the Tsawassen collection1 0COLLECTIONSITESOFAMBROSIACHAMISSONIS44 f- 0 ,which had as its primary minor compounds thiarubrine 7a and thiophene 7b. It is presumed thatthe thiarubrines are biosynthetically related although there has been very little investigation on thebiosynthesis of these compounds or acetylenic compounds in general.Figure 2.1 Collection Sites, in British Columbia, of Ambrosia chamissonisThe thiarubrines have very similar characteristic UV-Visible spectra. On the other hand,the thiophenes with their somewhat similar spectra, have distinguishing peaks (Appendix II).Therefore much can be gleaned from UV-Visible data of the latter. Because the thiarubrines arecolored compounds, column chromatography is useful for their isolation. Purification techniqueswere established for the thiarubrines and thiophenes using column chromatography for preliminaryisolation and then HPLC for final purification. HPLC was also employed for the evaluation ofcrude samples and column fractions. The HPLC was equipped with a photodiode array detectorwhich gives "in flight" UV-Visible spectra. A number of spectroscopic techniques were utilized toidentify the root chemicals: Ultraviolet-Visible spectrophotometry (UV-Vis), GasChromatography-Mass Spectrometry (GC-MS) of hydroxylated compounds, and solid probe MassSpectrometry (MS). Balza et al. (1989, 1990) have presented 1H NMR data in the structuralidentification of the compounds as well as assigned 13C NMR values. These techniques are notrequired for the known compounds since other spectral data being sufficient.MATERIALS AND METHODSA. Establishment ofExtraction, Isolation and Purification Procedures forThiarubrine and ThiophenesPlant MaterialRoots were collected from Centennial Beach, Tsawassen, British Columbia and transportedto the University of British Columbia in dark garbage bags to prevent photodegradation. Theywere immediately frozen to -70°C.Extraction for Developing Isolation and Purification TechniquesThe frozen roots were lyophilized, ground, and extracted over-night inmethanol:acetonitrile (7:3). The extract was filtered through Whatman 1 filter paper. Becausemuch of the thiarubrines is lost during the rest of the isolation procedure (Fig. 2.2) quantificationwas determined at this time. Quantification was accomplished using UV-Visiblespectrophotometry. The molar absorptivity (e) is the same for thiarubrine I a and 2 a (6=3000 at490 nm, e=10,300 at 340 nm). It is assumed to be the same for the other thiarubrines.Absorbance was measured at 490 nm, using the Beer-Lambert equation the overall thiarubrinecontent was calculated as follows:conc.(mg/m1)= Abs. x mol. wt. 12The extract was evaporated in vacuo at 28°C to almost dryness and resuspended inacetonitrile. This was filtered through glass wool to remove the precipitate. The sample was roto-evaporated to dryness and resuspended in acetonitrile and filtered. This step was repeated until thesolution was clear, ie., no further precipitation occurred.FREEZE-DRY PLANT SAMPLEGRIND AND EXTRACT 'WTTHCH3OH/CH3 CN (7:1)24 firsFILTEREVAPORATE UNDER REDUCED PRESSURE TO ALMOST DRYNESS,RESUSPEND IN CH3CNFILTER THROUGH GLASS WOOL, CONCENTRATE,AND RESUSPEND IN CHC13PARTITION BETWEEN CHC13/H20 UNTIL H20 LAYER IS COLORLESS,FOLLOWED BY DRYING OF ORGANIC LAYER WITH ANHYDROUS Mg504CONCENTRATETO A SMALL VORESUSPEND WITH HPLC^RESUSPEND IN CH2CGRADE AIETONITRILEHPLC (UV-VIS DETECTION) TOEVALUATE SAMPLE CONTAININGALL OF THE THIARUBRINESAND THIOPHENESHPLC (UV-VIS DETECTION),MASS SPEC, GC/MS ANALYSIS OF TMS1DERIVATIVES OF THIOPHENES HAVINGALCOHOL FUNCTIONAL GROUPSAND114 NMRFigure 2.2 Extraction Protocol for Thiarubrines and Thiophenes from Roots of Ambrosiachamissonis.13COLUMN CHROMATOGRAPHY(SILICA GEL 60,70-230 MESH)The extract was brought down to a small volume and then partitioned between chloroformand water. The chloroform layer was partitioned repeatedly with water until the aqueous layer wasclear. The organic layer was dried with anhydrous MgSO4, concentrated in vacuo, resuspended inHPLC grade acetonitrile, and filtered for HPLC. If long term storage was required the sample wassuspended in dichloromethane and stored in the dark in the refrigerator. Alternatively, freeze-driedmaterial was extracted initially for 24 hours with petroleum ether to gain the less polar thiarubrinesand thiophenes. This was followed by a 24 hour extraction with methanol.Evaluation of Crude Extracts with HPLCAnalysis by HPLC was done using a Waters HPLC with a photodiode array detector. Thiswas the instrument of choice because "in flight" UV-Visible spectra can be obtained which areuseful in the identification of the compounds. The extract was analysed by HPLC using apreparative MCH-10 (C18, 10 pm particles) column (4 x 300 mm), eluted with CH3CN:H20(72:28 and 50:50) at a flow rate of 1 mllmin in order to identify the thiophenes. A portion of theextract was irradiated to convert the thiarubrines to their corresponding thiophenes. Standards ofthiophenes lb, 2b, 3b, 4b, 5b, 6b, and 7 b, were co-injected with the extract to identify thecomponents and infer the structures of the corresponding thiarubrines. The thiosulphinate (9) wasidentified by its characteristic UV-Vis sped= (Appendix).Isolation of Thiarubrines and Thiophenes by Thin Layer and Column ChromatographyThin Layer Chromatography (TLC) was used to monitor extracts and fractions.Aluminum-backed silica plates (Aluminum-backed silica ge160 F254, 0.2mm) using two solventsystems: hexane:diethyl ether (9:1), and dichloromethane:acetone (30:1) were employed.Preparative TLC was employed using glass-backed silica ge16o F254 (2mm) withdichloromethane:acetone (30:1). A silca gel (70-230 mesh) column was prepared withdichloromethane (CH2C12) and loaded with the crude extract The column was eluted withCH2C12. When no more bands were eluted, acetone was added gradually. For large scale14separation a preliminary column (10 cm x 300 cm) was required to remove the more polarcomponents which remained at the top causing poor column performance. This was followed bychromatography of the collected red bands on a longer narrower column (5 cm x 500-1000 cm).In the case of thiarubrine 3 a, isolation was complicated by an accompanying oil, which could bereduced in volume by running the extract through a column numerous times.Purification and Identification of CompoundsPurification was accomplished by preparative HPLC using a Varian 5000 HPLC with aMCH-10 (8 mm x 300 mm) reverse phase steel column, eluted isocratically with H20:CH3CN at 3ml/min. The solvent mix was adjusted for each compound.Confirmation of the identities of the compounds was carried out by Mass Spectroscopy.The isolated hydroxylated thiophenes were converted to the trimethylsilyl (TMS) ethers by dryingthe sample (-1 mg) and then adding 10 ml of dry pyridine and 10 ml of N,0-bis-trimethylsilyltrifluoroacetarnide (BSTFA) at room temperature. GC-MS analysis was carried outon the TMS-derivatives using an automated Finnigan 1020 GC-MS equipped with a fused-silicacapillary column (30 mm x 0.25 mm) of SE-54 and operated in the El mode (70 eV) with an ion-source temperature of 95°C. Solid probe Mass Spectroscopy was also carried out with anautomated Finnigan 1020 GC-MS. The thiarubrines were identified by solid probe MS instead ofGC/MS because they are unstable at the high temperature required.B . Extraction in Spring and Autumn of Roots from Two Collecting SitesPlant CollectionsCollections were made from Centennial Beach, Tsawassen, British Columbia (Sept., June)and North Beach (at the end of Cemetery Road) in the Queen Charlotte Islands, British Columbia(Sept., June) . Three entire plants were taken from each site. Only one plant which had a differentleaf morphology (Type 2) was removed from North Beach due to a limited population of thevariety. All plants were placed in dark green garbage bags to prevent photodegradation of the15compounds. Five grams of ground freeze dried material was extracted, prepared and analysed byHPLC as already described.RESULTSThe tap roots from plants growing on Centennial Beach have well-developed periderm,with succulent lateral secondary roots lacking peridenn. The larger tap roots may be hollow due tonecrosis (Figure 23). Using longitudinal sections of the outer bark there are circular pocketswhereas closer to the center exhibit longitudinal strands, which are not necessarily connected. Thethiarubrines are located within canals in the outermost cortical region and immature peridermFigures 2.3 and 2.4).16cavitycanalsFigure 2.3 Localization of Thiarubrine Canals in woody roots of Ambrosia chamissonis (Cross-Section).Reverse phase HPLC (CH3CN:H20, 72:28 and 50:50) resolved the thiarubrines andthiophenes as shown in Figure 2.5. HPLC runs were made at 490 nm as well as at 340 nm(Figure 2.6) confirming that the compounds absorbing at the former wavelength were thiarubrinesthus justifying the calculations made for the quantification at this wavelength. "In flight" UV-Visible spectra indicated the peaks of the thiarubrines and the thiophenes, as well as another whichVisible spectra indicated the peaks of the thiarubrines and the thiophenes, as well as another whichrepresented an unknown polyyne, which will be referred to as "Unknown Polyyne #1",(Appendix III). When the solvent system was changed to a 1:1 mixture the thiarubrines and thethiophenes with polarity intermediate to thiarubrine 4 a and thiophene,  3 b, were resolved.Thiarubrines la and 2 a and thiophenes  lb and 2 b are not included in the figure because of theirlong retention times and the fact that their peaks become flattened and resolution is poor. Whenpeak enhancement was performed using standards of the thiophenes it became apparent that ahitherto unknown thiophene with a polarity intermediate to that of the primary alcohol and the diolwas present (Figure 2.5). The identification of this new compound and its correspondingthiarubrine (8 b and 8a respectively) will be presented in Chapter 3.Figure 2.4 Localization of Thiarubrine Canals in Non-Woody (Succulent) Roots of Ambrosiacharnissonis. (t = thiarubrine canal)17Figure 2.5 HPLC Trace at a Flow Rate of 1 ml/min at (i) 28:72 (H20:CH3CN) and (ii) 50:50(H20:CH3CN).18490340lanm3anmi^ ,^.J\0 10^20Retention Time in MinutesFigure 2.6 HPLC Trace of Crude Root extract at 340 nm and 490 nm at a Flow Rate of 1ml/min H20:CH3CN (28:72).19Table 2.1 Rf Values of the Thiophenes on Analytical Thin Layer Chromatography on Silica GelF254.CompoundHexane:Diethyl Ether(9:1)Dichloromethane:Acetone(30:1)lb 0.50 0.802b 0.50 0.803b 0.30 0.724b 0.00 0.105b - 0.516b - 0.557b - 0.40Solvent Mixture at 3m1/min..Extraction procedures using petroleum ether furnished only the less polar thiarubrines la,2a, and 3a, as well as very low concentrations of their corresponding thiophenes. Thecompounds are more stable in petroleum ether than methanol as indicated by lower concentrationsto the thiophenes. Methanol, however, readily extracted the more polar thiarubrines (4a, 5a, 6a,7a) as well as residual non-polar thiarubrines and thiophenes. A nonpolar compound was detectedat Rt = 27.33 (CH3CN:H20, 72:28, flow rate 1 inlknin). This represents the pentayneneaccording to the spectrum determined by Bohlmann eta!., 1973 (UV Amax nm 412, 382, 353, 330,285, 266).Preparative TLC was used to isolate thiophenes on a small scale. The band at Rf = 0.1 wasscraped off and extracted with CH3CN:CH3OH (7:1). White crystals precipitated. These weresubjected to GC-MS. It had a similar retention time to other sesquiterpene lactones which had beenidentified (Felipe Ba17a, personal communication), but the fragmentation pattern was different fromany sesquiterpene lactone known from Ambrosia chamissonis (Geissman et al., 1973) (seeAppendix V for mass spectrum)Column chromatography with dichloromethane was employed for initial isolation of allcompounds. HPLC analysis of the fractions before and after irradiation confirmed the retentiontimes of the thiarubrines and their corresponding thiophenes. UV-Vis spectra of thiophenes20furnished by the HPLC photodiode array detector are shown in Appendix H. The thiarubrines tendto degrade and therefore gave less defined spots. Thiarubrine 1 a and 2a came off together as thefirst red band followed by thiarubrine 3a. Two red bands eluted next. The first one was thealready identified primary alcohol 7a as monitored by HPLC co-injected with a standard. HPLCof crude samples revealed that there was another polyyne which eluted at the same retention timecausing additional peaks in the UV-Visible spectrum. This was removed with columnchromatography. The chlorohydrins were not visualized but were detected by HPLC. Theyappeared in fractions between the primary alcohol 7a and the epoxide 3a. A band closely followed7a, whose identification will be described in Chapter 3. Thiarubrine 4 a eluted only after acetonewas added to eluting solvent. The thiophenes co-chromatographed with their correspondingthiarubrines.Preparative HPLC conditions were varied depending on the polarity of the thiarubrines andthiophenes to be separated. The retention times and solvent systems are presented in Table 1.3.for the thiarubrines and thiophenes. The purification of thiarubrines la and 2 a and the thiophenes1 b and 2 b was difficult because they could not be separated on the column and thus could only beseparated if lower concentrations were injected at a time on HPLC (-2 mg), whereas the others canbe loaded 8- 10 mg at a time. Mass spectral data of a mixture of l a and 2a is presented in Figure2.7. When a crude or pure thiarubrine solution is irradiated it turns yellow. When the resultingthiophene is isolated by HPLC the pure compound is colorless.21Table 2.2 Analytical HPLC Retention Times (in Minutes) of the Thiarubrines and ThiophenesWith Different Solvent Systems (H20:CH3CN).Compound 28:72 50:50 55:45la 18.62 - -lb 15.66 -2a 17.52 - -2b 14.63 - -3a 11.72 69.52 -3b 9.99 52.09 -4a 4.88 12.13 17.864b 4.37 9.46 13.335a (8.39) 39.50 71.325b (7.50) 30.10 52.086a (7.26) 34.74 62.586b (6.36) 26.07 45.427a (8.05) 35.35 58.007b (6.91) 25.15 41.598a (8.05) 30.31 51.008b (6.91) 23.02 37.509 - 27.73 43.02"Polyyne #1" 13.40 - -values in brackets indicate that at this solvent system the peaks are not ressolved22Table 2.3 Preparative HPLC Retention Times (in Minutes) for the Purification of Thiarubrinesand Thiophenes with Different Solvent Systems.Compound II2 0:CH3C N Rt(min)^Il a 35/65 41.82lb 35/65 33.962a 35165 39.022b 35/65 31.463a 35/65 22.593h 35/65 18.724a 60/40 42.864b 60/40 29.075a 50/50 42.775b 50/50 31.826a 50/50 38.946b 50150 26.947a 50150 38.467b 50/50 29.40*Solvent Mixture at 3m1/min, Rt = Retention timeAlthough relative retention times and UV spectra are known for some of the thiophenesmass spectral data substantiated the identities of these compounds. The hydroxylated thiophenessuch as the chlorohydrin thiophenes (5 b and 6 b) in Figure 2.8, the primary alcohol thiophene(7b) in Figure 2.8 (ii) and the diol (4 b) in Figure 2.9 (i) were identified using GC-MS of the TMSderivatives. The chlorohydrins have very distinctive fragmentation patterns which distinguish onefrom the other (Figure 2.10). Being chlorinated compounds they have very distinct spectra due tothe relative abundances of the chlorine isotopes. Solid probe was used for the thiarubrines.Thiarubrine 4 a for instance, crystallized in concentrated solution from preparative HPLC. Thecrystals were subjected to solid probe MS revealing the characteristic mass spectrum (Figure2.10 ii).23Figure 2.7 Mass Chromatogram and Spectrum of a Mixture of Thiarubrines 1 a and 2 a Isolatedby Column Chromatography.24Figure 2.8 (i) GC-Mass Chromatogram of the TMS-Derivatives of Thiophenes 4 b, 5 b, 6 b, 7 b,8 b. (ii) Mass Spectrum of the TMS-Derivative of Thiophene 7 b.25Figure 2.9 (i) GC-Mass Spectrum of the TMS-Derivative of Thiophene 4b and (ii) Solid ProbeMass Spectrum of Thiarubrine 4a Crystallized from Preparative HPLC.26Figure 2.10 GC-Mass Spectra of the TMS-Derivatives of Chlorinated Thiophenes 5 b and 6 b.2 710^20^1 0^20Time in MinutesFigure 2.11 HPLC Traces of Roots Collected from Plants with Two Types of Leaf Morphology28 CB-S (AC)CB-W(AC)Q-1 (AC)CB-S (SC)CB-W(SC)Q-1(SC)Q-2(SC)0.00^0.25^0.50 0.75^1.00^1.25^1.50^1.75^2.00^2.25^2.50Thiarubrine Content in mg/g. dry wt.Figure 2.12 Thiarubrine Content of Collections from Centennial Beach (CB) (S=Succulent ,W=Woody) and the Queen Charlotte Islands (Q) in the Spring (SC) and Autumn (AC). Variationis presented as standard deviation (n=3).CB-S(SC) CB-W(SC) 0-1 (SC) CB-S(AC) CB-W(AC) 0-1 (AC) 0-2(AC)1008 06 04 0 -2 0 -0The HPLC traces of the roots from the collections of Ambrosia chamissonis from theQueen Charlotte Islands demonstrated different thiarubrine profiles (Figure 2.11). The chemicalprofile of Type 2 were very similar to those from Centennial Beach which were similar in leafmorphology to Type I. Type 1 exhibited the highest yield of thiarubrines on a thy weight basis(Figure 2.12).Collection SitesFigure 2.13 Thiarubrine Profiles of Collections from Centennial Beach (CB) (S=Succulent ,W=Woody) and the Queen Charlotte Islands (Q-1 and Q-2, representing Type-1 and Type-2 leafmorphologies) in the Spring (SC) and Autumn (AC).The chemical profile of the succulent roots did not vary with season in the Tsawassensamples, whereas the woody roots did. The latter had much higher levels of thiarubrine 3a in theautumn than in the spring. The converse was true for thiarubrine 4 a.The roots of the QCI 1 and QCI 2 collections did not differ in appearance. They both hadvery little in the way of succulent or lateral branching, and were generally much smaller than thosefrom Centennial Beach. The roots of QCI-1 had very high levels of thiarubrine 4 a, whereas QCI-2 had a very similar profile to CB-S. Examination of HPLC traces of the minor thiarubrines andthiophenes revealed that the primary alcohols 7a, 7 b, 8 a, and 8 b were present in appreciable29concentrations (-4% of the total thiarubrineithiophene content). The chlorohydrins were detectableby "in flight" UV-Vis spectra in some cases, but often they were not integrated by the HPLC.DISCUSSIONThe roots of Ambrosia chamissonis produce the widest spectrum of thiarubrines andthiophenes known from any other species in the Asteraceae. They are all based on the structure ofthiarubrine la with the exception of thiarubrine 2a. Figure 2.14 demonstrates the proposedbiosynthetic relationship between some of the thiarubrines. Extraction and purification procedureshave been established to obtain the compounds with their differing polarities. It may be that otherspecies examined have a wider array of chemicals which have not been detected due to theextraction procedures used. Column chromatography was useful for the isolation of the minorthiarubirnes and thiophenes, which could be analyzed by GC-MS. The low production of thesecompounds implies that their defensive function in the plant may be minimal. The presence ofchlorohydrins may reflect the abundance of chlorine ions in the habitat of this species.Chlorohydrins can be artifactually derived from epoxides, but the relative abundances of theprimary and secondary chlorohydrins suggest that this is not occurring. If they were artifacts theprimary chlorohydrin would be three times the abundance of the secondary. HPLC traces indicatethat the converse is true of extracts from roots and root cultures of Ambrosia chamissonis.Differences in plant morphology were observed in and between two populations. Theobservations of Geissman et aL(1973) that correlation cannot be made between leaf morphologyand chemistry hold true for those samples examined from the Queen Charlotte Islands andTsawassen. The plants which had similar leaf morphology had very different chemical profiles,whereas plants that were different (CB-S and QCI-2) had very similar profiles. The morphologicaltypes as well are not steadfast; some plants observed had varying leaf morphology within oneindividual. Although there is variability in both chemistry and morphology the chemicals present3031are unique to this group. These compounds may be chemical markers in the identification ofspecies in these groups of plants which are troublesome taxonomically.Nothing is known about how the plant responds to the sulphur that is extruded from thethiarubrine molecule. The yellow coloration of the thiarubrine extract after irradiation is probablydue to the sulphur since the thiophenes are colorless. Other sulphur forms such as sulphite andsulphur dioxide are normally converted into sulphate which can be stored in the cell vacuole(Rennenberg, 1984). It is this form which is reduced and subsequently utilized by the plant. Inthe case of thiarubrines, which are stored in canals, the canals may be a storage place for thesulphur as well. The canals do not only contain thiarubrines, thiophenes, and their byproducts;Throughout the many extractions and attempts at isolation there is an oil in which these substancesare dissolved. Nothing is known about its chemical nature.Figure 2.14 Proposed Biosynthetic Relationship Between some of the Thiarubrines.CHAPTER THREECOMPARISON OF THIARUBRINE AND THIOPHENE PRODUCTIONBETWEEN ROOTS AND ABOVE GROUND PORTIONS OFAMBROSIA CHAMISSON1SINTRODUCTIONSpecies in the Asteraceae, such as Chvenactis douglasii (Cosio eta!., 1985) and Verbesinassp. (Bohlmann eta]., 1980), contain no thiarubrines or thiophenes in their above groundportions. Roots are generally considered the major sources of these classes of compounds. Thethiarubrines are unstable in light and both thiarubrines and thiophenes are bioactive uponirradiation. Theoretically organs exposed to sunlight seem to be suitable for storage because ofpossible photo-auto-toxicity. There are plants, however, which do store photo-unstable polyynes,such as phenylheptatryine, in their leaves.The leaves of the plants collected at Centennial beach in Tsawwasen, B.C. exhibited littlevariation in morphology as indicated in Chapter Two. In this chapter the distribution ofthiarubrines and thiophenes will be examined in the entire plant Quantitative HPLC data wasadjusted to take into account the differences in absorptivity by the two classes of compounds.Thus the peaks on the HPLC reflect the amounts of compounds based on their absorptivity at theparticular wavelength. At 340 nm the molar absorptivity of thiarubinre 1 a is 10,000, whereas thatof thiophene 1 b is 31,000.The location of thiophenes has not been determined accurately. X-ray emmissiontechniques have been employed to determine the sulphur distribution in Tagetes patula seedlingroots (Makjanic eta!., 1988). The lowest concentration was found to be in the epidermis whereasthe highest was found to be in the endodermis. The secondary roots of A. chamissonis have awell-developed bark. This means that the endodermis is lost, since the pericycle, the layer internal32to the endoderm, develops into the periderm The localization of these compounds has not beenexamined in other parts of the plant. Unlike the colourless thiophenes, the red thiarubrines areeasy to visualize.MATERIAL AND METHODSPlant MaterialCollections of Ambrosia chamissonis were obtained from Centennial Beach, Tsawwassen,British Columbia October 8, 1989. Three plants with thick succulent stems were selected (ie.shoots without periderm). The entire plants were placed in dark plastic garbage bags to minimizethe exposure to light..Histological ExaminationSectioning using resin embedding techiniques (JB-4) was not succuessful due to pigmentdegradation under the conditions of fixing and embedding. Hand-sectioning in dim light gavemuch better results. Roots, stems and leaves of each sample were hand-sectioned to determine thepresence and localization of the red compounds.ExtractionLeaves (7-10 cm long), green stems, woody stems, and roots were separated and samplesof approximately 20 grams of each were weighed out. All samples were frozen at -70°C andfreeze-dried. Five grams of each sample were weighed and ground and extracted overnight inmethanol:acetonitrile (7:1). The solution was filtered through Whatmann No. 1. The thiarubrineconcentration of each extract was determined using the Beer-Lambert equation for the roots andwoody stems. The thiarubrine content of the green stems and leaf extract could not be determinedat this stage because of other pigments which absorb at the wavelengths used for calculations ofconcentration.33Evaluation of Extracts with HPLCThe extract was brought down to a small volume and then partitioned between chloroformand water. The chloroform layer was partitioned repeatedly with water until the aqueous layer wasclear. The organic layer was dried with anhydrous MgSO4, concentrated in vacuo, resuspended inHPLC grade acetonitrile, and filtered for HPLC. Analysis by HPLC was done using a WatersHPLC with a photodiode array detector. Examination of all extracts was performed by HPLCusing a Waters System with a MCH-10 column (4 mm x 300 mm) at a flow rate of 1 ml/mineluting isocratically with CH3CN:H20 (72:28). All runs were performed at 340 nm except forsome of the leaf extracts which were also analyzed at 484 nm. All samples were irradiated andthen analyzed by HPLC to verify that the peaks were thiophenes and thiarubrines. All otherprocedures were carried out in dim light to prevent the photo-conversion of thiarubrines tothiophenes. Calculations used peak area percentages from HPLC traces. The thiophene valueswere adjusted by dividing the percent by 3.1 and then overall thiarubrine and thiophene levels werecalculated as a percent fom the total.Column ChromatographyHalf of each of the three extracts of the green stem and the leaf were combined. Theextracts were separated by column (5cm x 45cm) with a cold water jacket packed with silica gel(230-400 mesh) and hexane:diethyl ether (9:1). The stem and leaf extracts were loaded onto thecolumn and chromatographed with the same solvent mixture. A red band (thiarubrines) wascollected and the overall thiarubrine content was calculated. The fraction was analyzed by HPLCto ensure that all of the thiarubrines wer present in the fraction.34RESULTSIn the spring young shoots have conspicuous red stripe which upon extraction reveal thatthey are thiarubrines and anthocyanins. The thiarubrines can be seen in canals runninglongitudinally in the upper stem and around the hollow stem of the lower shoot (Figure 3.1).Mature plants were also examined chemically.Figure 3.1 Thiarubrine Canals in the Young Stem (i) and Leaf (ii) of Ambrosia chamissonis.In general leaves and stems of young tissues, contain more thiarubrines than older, largerleaves. Woody stems also contain thiarubrines, but are impossible to discern histologically using35the present method because of dark brown pigmentation and the brittleness of this material.Extraction revealed the presence of thiarubrines and thiophenes.Table 3.1 Overall Thiarubrine Content of the Different Organs of Ambrosia chamissonisPlant OrganOverall Thiarubrine Content(mg/g dry wt.)Roots 2.1 ± 0.021Woody Stems 0.234 ± 0.040Green Stems 0.102Leaves 0.166Error is expressed as Standard Deviation (n=3), no error was given for leaves andgreen stems because these samples were combinedPreliminary HPLC analysis indicated that the thiarubrine in highest abundance was la.Methanolic extracts were partitioned with petroleum ether to separate the more polar components.Column chromatography successfully separated the thiarubrines from the other pigments found inthe leaves. An orange band was conspicuous which separated into a fast yellow band and a slowerred band. The yellow band was collected and the UV-Vis spectrum was examined (Amax 343 nm,452 nm, 477 nm) which is characteristic of carotenoids. The red band split into a yellow and a redone. These fractions were not used for calculations of content because much of the thiophene islost during the process. The UV-Vis spectrum of the former indicated this fraction containedthiophenes, whereas the latter contained a mixture of thiarubrines and thiophenes. Increasing theproportion of hexane in the eluting solvent eluted a green band, which gave a UV-Vis spectrumtypical for chlorophyll (Xmax 664, 437).The root profile is of course similar to the extracts examined in Chapter Two. There wasvariation in the thiarubrine 3a content as is reflected by the large error bars of thiarbrines 3a andla (Figure 3.2). There is a very low thiophene content. The chemical profile of the woody stems(Figure 3.3) mimicked that of the roots except that there was a higher ratio of thiophenes to36la^lb^2a^2b^3a^3b^4a^4bCompound37Figure 3.2 Percent of Thiarubrines (1a, 2a, 3a, 4a, 7a, 8a) and Thiophenes (lb, 2 b, 3 b, 4 b,7b, 8 b) from the Total Polyyne Content of the Root Extracts. Standard Deviation is Expressed inError Bars (n=3). (P 1=Unknown Polyyne #1)la lb 2a 2b 3a 3b 4a 4b 7a18a7b18b P 1CompoundsFigure 3.3 Percent of Thiarubrines (1a, 2a, 3a, 4a, 7a, 8a) and Thiophenes (lb, 2 b, 3 b, 4b,7 b, 8 b) from the Total Polyyne Content of the Woody Stem Extracts. Standard Deviation isExpressed in Error Bars (n=3). (P 1=Unknown Polyyne #1)1 0 -„v,771 a^1 b7-77'2a^2b^3a^3b^4a^4b 7a/8a7b/8b P 1CompoundsFigure 3.4 Cross-section Through Green Stem with Thiarubrine Canals of Ambrosiachamissonis. (t = thianibrine canal)igure 3.4 Percent of Thiarubrines (1a, 2 a, 3 a,  4 a, 7 a, 8a) and Thiophenes (lb, 2 b, 3 b, 4 b,'b, 8 b) from the Total Polyyne Content of the Crude Green Stern Extracts. Standard Deviation isxpressed in Error Bars (n=3). (P 1=Unknown Polyyne #1)3874'-12a^2b^3a^3b^4a^4b 7a/8a7b/8b P 1Compoundsla^lb39Figure 3.6 Cross-Section Through the Leaf, with Thiarubrine Canal, of Ambrosia chamissonis.(t = thiarubrine canal)to 40a)(• 30s-40 20_so10Figure 17 Percent of Thiarubrines  (1 a, 2a, 3a, 4a, 7a, 8a) and Thiophenes (lb, 2b, 3b, 4b,7 b, 8 b) from the Total Polyyne Content of the Crude Leaf Extracts. Standard Deviation isExpressed in Error Bars (n=3). (P 1=Unknown Polyyne #1)thiarubrines (approximately 1:1 as estimated). There was also a very high concentration of 2 bexceeding that of the other thiophenes including thiophene la. Column chromatography was notrequired for the visualization of the polyynes in woody stems because there were no otherpigments absorbing at 490 nm.The green stems and leaves have a very high thiophene to thiarubrine level (Figures 3.5 and3.7), but as the TLC and visual inspection indicate thiarubrines are present (Figures 3.4 and 3.6).The former have a higher content of thiarubrine 4a than the latter in which the level is negligible.In all samples the minor thiarubrines and thiophenes discussed in Chapter Two weredetected. The chlorohydrins were in negligible amounts whereas the primary alcohols 7a, 7 b, 8a,and 8 b could be quantified. They were in small quantities, therefore their values were combined.The thiophene content relative to the thiarubrines was 1:3 in the aerial parts and 3:1 in root extracts.A root extract was column chromatographed under the same conditions as the leaf andgreen stem extracts. Five percent of the yield was lost in this procedure, and the chemical profiledemonstrated a lower thiophene content.DISCUSSIONThiophenes have been shown to have an independent biosynthesis to the thiarubrines(Constabel and Towers, 1989b). In the case of the leaves where there is a high concentration ofthiophene relative to thiarubrine presumably much of the former is derived through the degradationof the latter in the shoots and leaves. Thiophene 2 b is in much higher concentrations in the woodystems than would be predicted for normal degradation when compared with the other thiophenes.This may be the result of differential stability of this molecule or it may be due to elevatedsynthesis of thiophene 2 b independent of the thiarubirne 2a.The thiarubrines, although considered root chemicals, are also found in other portions ofthe plant, albeit in lower concentrations. These chemicals are powerful phototoxins whenirradiated yet surprisingly they are present in the leaves and stems. The canals are very important40for the maintenance of the compounds and the protection of the plant from the chemicals it issynthesizing. They are especially noticeable in young tissues and presumably function asdefensive sinks. How the plant maintains them is not known. Defensively it is a good strategy;exposure by the invading organism to light after consumption leads to its instantaneousdestruction. Why should a phototoxic chemical occur in the roots if predation is in the dark? Onepossible reason is that the predator may surface after consumption and thus be effected at a latertime. An important feature of the thiaubrines is their light and dark reactions. There is someevidence also that thiophenes, such as a-terthienyl, can be electronically excited, in the absence oflight, as a result of enzymatic reactions which produce singlet oxygen or superoxide anions.Peroxidase activity in Tagetes roots increased with plants infected with nematodes (Gommer et a!.,1988). Thus we have the same reaction that would occur photochemically.41CHAPTER FOURIDENTIFICATION OF A NOVEL THIARUBRINE AND THIOPHENEINTRODUCTIONAs indicated in Chapter Two, reverse phase HPLC revealed a novel thiarubrine andthiophene in A. chamissonis roots. Detection by UV-Vis spectrophotometry is important becausemost of the polyacetylenes exhibit characteristic spectra. To identify these compounds a number ofchromatographic techniques such as preparative thin layer chromatography, high performanceliquid chromatography are employed. Mass spectrometry is an important tool for the elucidation ofchemical structures. Not only does it supply the molecular formula, but the fragmentation patternsalso give an indication of structure. The fragmentation patterns of the chlorohydrins 5 b and 6 b,for example, are clearly distinguishable (Balza et al., 1990, Chapter 1). Gas chromatography-mass spectroscopy (GC-MS), of the TMS derivatives, can be employed when examining mixturesof alcohols and acid derivatives of thiophenes, but thiarubrines are not stable enough at the hightemperatures required for this procedure. Information about the thiarubrines can be acquired usingsolid probe electron impact mass spectrometry.As already mentioned 13C-NMR spectroscopy has not been used very much for structuralinformation of these compounds, and by far the most useful technique for the elucidation of thechemical structures of both a thiarubrine and its corresponding thiophene is 1H-NMR spectroscopywhich allows for characterization of the protons in a compound. The characteristic shifts andcouplings of thiophene and thiarubrine protons make interpretation of data rather simple. ProtonNMR data for several thiophenes was presented by Bohlmann and Zdero (1985). They indicatedthat the acetylenic methyl groups is always at lower fields at around 2.05 ppm. The long-rangedeshielding effect of the ring protons are also characteristic.4 2MATERIALS AND METHODSPlant Material.Ambrosia charnissonis (Less.) Greene was collected in British Columbia at CentennialBeach (CB), Tsawwassen, British Columbia in May and September, 1991. Voucher specimensare deposited at the herbarium of the Department of Botany, University of British Columbia.Extraction,.Freeze-dried ground roots were extracted with CH3OH:CH3CN (7:1) for 24 hours. Thesample was filtered through Whatman 1 filter paper and evaporated in vacuo to almost dryness thenresuspended in CH3CN. The volume was reduced and partitioned at least twice between CHC13and 1420. The organic layer yielded the thiarubrines. Preliminary examination of all extracts andfractions were performed by HPLC using a Waters System with a MCH-10 column (4 mm x 300mm) at a flow rate of 1 ml/min eluting isocratically with CH3CN:H20 (72:28). All procedureswere carried out in dim light to prevent the photo-conversion of thiarubrines to thiophenes.Isolation and Identification.Preliminary separation of 8a was carried out using a Si gel 60 column (230-400 mesh)eluted with CH2C12 and monitored by analytical HPLC (see Chapter Two). Compound 8a wasfurther purified by preparative HPLC using a Varian 5000 with a MCH-10 (8 mm x 300 mm)reverse phase column, eluting isocratically with H20:CH3CN (1:1) at 3 ml/min. High and lowresolution electron impact mass spectral analysis was carried out at the University of BritishColumbia Mass Spectrometry Facility.For the identification of the thiophenes a small amount of crude red oil obtained from thecolumn and determined by HPLC to contain thiarubrine 8 a was irradiated to produce thecorresponding thiophenes. Preparative TLC (CH2C12:CH3COCH3, 30:1) yielded a mixture ofthiophenes 7 b and 8b (RI 0.28). These were converted to the trimethylsilyl (TMS) ethers by4 3drying the sample and then adding 10 ml of dry pyridine and 10 ml of N,0-bis-trimethylsilyltrifluoroacetamide (BSTFA) at room temperature. GC-MS analysis was carried outon the TMS-derivatives using an automated Finnigan 1020 GC-MS equipped with a fused-silicacapillary column (30 mm x 0.25 mm) of SE-54 and operated in the El mode (70 eV) with an ion-source temperature of 95°C. GC-MS was carried out on a mixture of the hydroxylated thiophenesto determine their relative retention times. 8 a and 8 b were purified by preparative HPLC andsubmitted for 1H NMR spectroscopic analysis.RESULTSAnalytical reversed-phase HPLC analyses with CH3CN:H20 (1:1), revealed that 8 a(Rt=30.3 min) has an intermediate polarity between 7a (Rt=35.4 min) and its correspondingthiophene (Rt=25.2 min), whereas the thiophene 8 b (Rt=23.0 min) has a slightly lower polaritythan the thiophene 7 b (Rt=25.2 min). Figure 4.1 shows the UV-Vis spectra of thiophenes 8 b and7b. The UV-Vis spectrum of thiarubrine 8 a is presented in Figure 4.2. Preparative HPLC wasuseful in the purification of thiarubrine 8 a and thiophene 8 b. The optimum conditions were a flowrate of 3 ml/min with CH3CN:H20 (1:1). Thiarubrine 8a had a retention time of 35.17 minwhereas thiophene 8 b was eluted after 26.60 min.Compound 8 a was determined to have a molecular formula of C131110S20 (M;246.0174) by high resolution El mass spectrometry (Figure 4.3). Other major fragments includedmh 214, suggesting the loss of a sulphur atom and [M-CH20Hr (m/z 215) which ischaracteristic of a primary alcohol. The UV-Vis spectrum of 8 a is very similar to those of theother thiarubrines, whereas the absorption spectrum of the corresponding thiophene (8 b) isdistinctive, resembling that of 4b and 5b in having an additional UV maximum which is indicativeof an alcohol functionality. The conversion of thiarubrines to thiophenes was carried out byirradiation of the red fractions, which turned yellow. Upon purification by HPLC the thiophene8 b was obtained as a colorless solution.4451112J.._1_,...1^1-•-■ ---■^.I^-I^' •--I -,^IHPLC Trace (Retention Time in Minutes)7b8b45Figure 4.1 "In Flight" UV-Visible Spectra of Thiophene 7 b and 8 b from HPLC.The GC-MS analysis of the trimethylsilyl ether derivatives of the sample isolated from TLCfurnished two peaks corresponding to a molecular ion at m/z 286 (thiophene 8 b) and a molecularion at m/z 284 (thiophene 7b) (Figure 4.4). The mass spectrum of the former (Figure 4.5), andsubsequent analysis of samples purified by HPLC, revealed major peaks at [M-CH2OTMSr (m/z183) and [CH2OTMS] (m/z 103) indicating the presence of a primary hydroxyl group. The GC-MS analysis of a mixture of the hydroxylated thiophenes from Ambrosia chamissonis (4 b, 5 b,6 b, 7 b, and 8 b) demonstrated their relative retention times (Figure 4.6).Figure 4.2 UV-Visible Spectrum of Thiarubrine 8 a.4 6Figure 4.3 High Resolution Mass Spectrum of Thiarubrine 8a.Figure 4.4 GC-Mass Chromatogram Of Sample Isolated by TLC.The 300 MHz 1H NMR spectrum of the thiophene 8b (Figure 4.7.) showed a signalcorresponding to an acetylenic methyl singlet at 2.07 ppm and an AB doublet of doublets at 7.10ppm (H-8) and 6.93 ppm (1-1-9). Resolution enhancement showed a long-range coupling(6J058) between the latter (H-9) and the methyl group, which aided in the identification of thering proton of the thiophene moiety. An oxygen-bearing methylene at 3.86 ppm (t, J=6.22, 6.30),coupled to a methylene at 2.66 ppm (t, 2H, J=6.22, 6.30), typical of protons of a carbon atombonded to a carbinol and acetylenic groups, was also observed. Irradiation of the signal at 3.86ppm collapsed that at 2.65 ppm to a singlet, inferring a primary alcohol function in the molecule.The 1H NMR (300 MHz) spectrum of 8 a revealed similar resonances to that of the thiophene. Theacetylenic methyl signal was found at 2.06 ppm (s, 3H), and the ring proton resonances at 6.64ppm (H-8) and 6.49 ppm (H-9). As above, a long-range coupling (6J=0.79) was also observed47Figure 4.5 GC Mass Spectrum of Thiophene 8 b and Fragmentation Pattern of the Molecule.4849Figure 4.6 GC Mass Chromatogram of the TMS Derivatives of the Hydroxylatecl Thiophenes.(A = TMS derivative of thiophene 7 b, B = TMS derivative of thiophene 8 b, C = TMS derivative of thiophene 6 b,D = T1VIS derivative of thiophene 5 b, E = 'TMS derivative of thiophene 3 b)between the acetylenic methyl group and the H-9 proton, which afforded ready distinction betweenthe six-membered ring protons signals. Thiarubrine 8a was thus characterized as 3-(1-propyny1)-6-(6-hydroxyhex-3-yn- 1 yny1)- 1 ,2-dithiacyclohexadiene and the thiophene 8 b as 2-( 1 -propyny1)-6-(6-h ydroxyhex-3 -yn- 1 yny1)-th iophene.Figure 4.7 300 mHz 1H NMR of Thiophene 8b.DISCUSSIONThese results bring to date the presence of eight thiarubrines, and their correspondingthiophenes, in Ambrosia chamissonis roots. UV-Vis spectra are very useful in the diagnosis of the50thiophenes, although the UV-Vis spectrum of the new secondary alcohol was somewhatmisleading. Based on similar molecules, the extra absorbance peak (341 nm) suggested, that thehydroxyl group was probably adjacent to an acetylene group, implying that the compound is asecondary alcohol (Felipe Balza, dir. comm.). This was ressolved by derivatizing the hydroxylgroup followed by GC-MS. This fragmentation pattern furnished the position of the hydroxylfunction. The thiarubrine does not lend itself well to GC-MS analysis due to the high temperaturesrequired. Low and high resolution solid probe electron impact mass spectrometry of the purethiarubrine however, supplied the required information. By far the most powerful technique forthe elucidation of the structure of the thiophene and thiarubrine was 1H NMR spectroscopy. Itreadily allows for the distinction between positional isomers; thiarubrines based on the 1 a skeletonexhibit coupling between the ring proton (H-9) and the acetylenic methyl group, whereas thosebased on the 2 a skeleton do not exhibit coupling at all between these two moieties.Thiarubrine 8 a is presumably biosynthesized through thiarubrine 3a via hydrogenation toproduce the secondary alcohol There is no evidence of the presence of the related primaryalcohol.51CHAPTER FIVEESTABLISHMENT OF ROOT CULTURESINTRODUCTIONTissue culture is potentially a good alternative source of valuable phytochemicals. Theproblems confronting this technology include: low yields of chemicals, problems withestablishment of continuous cultures, and the fact that many desired compounds are rarely exudedinto the medium. The production of the naphthoquinone pigment shikonin, an importantcompound in the cosmetic industry for coloring lipstick and in the medical field for the treatment ofburns and hemorrhoids, is one of the few examples of industrial utilization of plant tissue culturefor the gain of chemicals (Fujita et aL, 1981). A two stage system using suspension culture ofLithospermum erythrorhizon is utilized to produce shikonin. In the first stage cells proliferate in adefined medium whereas the second stAge medium is modified for chemical production.Goleniowski et aL(1990) examined the sequiterpene lactone production in callus culture ofAnibrosia tenuifolia. They claimed that levels of these compounds were higher than in the wholeplant growing under natural conditions. Preliminary studies reveal, however, that thiarubrines arenot produced in callus cultures of Ambrosia chamissonis. There are many examples which showthat for production of certain secondary compounds some degree of organization is required (Cosioet al., 1986). Roots of Ambrosia chamissonis are the best source of thiarubrines and thiophenes,as has been demonstrated in previous chapters, thus root cultures were examined. Roots are easilyinduced through modification of growth regulators in the medium, such as the use of a higher ratioof auxin to cytokinin which characteristically favours the formation of roots.Another way to propagate roots in culture is to employ Agrobacterium rhizogenes. Anexcellent review article is presented by Zambryski et al. (1989). This gram-negative plantpathogen causes hairy root disease so-called because it induces the proliferation of roots at the site52of infection. The mode of activity is much the same as for A. tumeAciens. Agrobacterium specieshave plasmids called root-inducing (Ri) plasmids in the case of A. rhizogenes and the tumour-inducing (Ti) plasmids in A. tumefaciens. The bacterium introduces a piece of DNA from the Ri-plasmid, known as transfer DNA (T-DNA), into a wounded plant cell where it becomes stablyincorporated into the nuclear genome (Figure 5.1). Vir genes, on the plasmid, facilitate thistransfer. Factors released from the plant cell upon wounding enhance the expression of thesegenes; for example, acetosyringone from tobacco, has been identified as a virulence inducer.Figure 5.1 Transformation of Ambrosia chamissonis with Agrobacteriu rhizogenes.Among other things, the T-DNA encodes for the synthesis of opines. The Ri-plasmidcontains genes, which not only encode for the replication and transportation of the T-DNA, butalso encode for the metabolism of opines. The plant cell is thus reprogrammed not only for theproliferation of tissue, but also for the production of a carbon and nitrogen source for the bacteriawhich is not metabolizable by the plant. There are two main types of A. rhizogenes strains basedon the types of opines encoded for. The most common is the agropine-type characterized byhaving two T-DNAs. Put simply, one of them (TR-DNA) encodes for the production of auxin.The TL-DNA sensitizes the plant tissue to endogenous auxin by virtue of root loci (rol) genes. The53mannopine-type has one T-DNA which has high homology with the TL-DNA. Transformed rootsare therefore the result of increased sensitivity to endogenous auxin levels.A. tumefaciens, in particular, has been used as a "natural" genetic engineer, making itpossible to incorporate foreign DNA into plant cells which can then be regenerated into entireplants. Regenerated plants from hairy roots have characteristic morphologies due to the rol genes.These features include wrinkled leaves, stunted growth and decrease in seed output. Theapplication of A. rhizogenes ito tissue culture is slightly different than that with A. tumefaciens.The latter has been extensively used in genetic manipulation and engineering, whereas the former ismainly used as a source of phytochemicals.Tissue cultures are established by introducing A. rhizogenes to wounded plant tissue, thetransformed cells developing into roots. These roots can be maintained on media devoid of growthregulators, therefore modification of the medium is not required. The morphology of transgenicroots is different from that of normal root cultures. They grow ageotropically ie. they growupward, away from gravity and are thus easily recognized. Proof of transformation is establishedin a number of ways. High voltage paper electrophoresis is commonly used as a "simple"separation technique for the detection of the opines. Over time some cultures may lose their abilityto produce these compounds and a negative result may be obtained. A number of researchers haveused DNA probes to detect the incorporated T-DNA.The suspension culture system already described for shikonin production, has not beenwithout its problems. Due to the genetic instability of non-differentiated tissue, Shimomura a al(1991a) examined the production of shikonin in hairy-root cultures. They were able to extract thiscompound from the medium without changing the culture conditions. The production wasenhanced by using an adsorbent (XAD-2 column) and 5.2 mg per day could be obtained from a 2-liter batch.Constabel and Towers (1988) developed hairy root cultures of Chaenactis douglasii andexamined the production of thiarubrines. Through selection of high-yielding lines they were ableto obtain cultures which produced twice as much thiarubrine as non-transformed root cultures.54Shimomura et a/. (1990b) suggest that since hairy root cultures of Hyoscyamus, generated fromtwo different strains, exhibited different chemical profiles; it is the insertion of different "plasmids"which is responsible. They did not specify how many lines they examined, however, so it may bea matter of selection of a higher yielding line. Mano et aL (1986) indicate that lines from the samestrain show variations in chemical profile.Mukundan and Hjortso (1990) studied the production of thiophenes in root cultures ofTagetes erecta. Like many researchers they found that the overall production of thiophenes innormal root cultures versus hairy root culture was less. This was calculated on the basis of theyield per flask rather than the amount of thiophene per gram of root material. The chemical profileof the transformed roots matched that of the intact roots. This was also observed by Parodi et al.(1988) in a study of the bithiophenes and benzofurams of hairy root cultures of Tagetes patula.Efforts have been made to up-scale production of desired chemicals through the use offermenters eg. shikonin production in suspension cultures of Lithospermum. Hilton and Rhodes(1990) found problems with growing root cultures using the same style of fermenter. Theimpellers of the culture vessel, which keep the culture and medium circulating, ensuring adequateaeration, caused damage to the roots. This problem was solved through modification of the vesselby segregating the impeller with a cage. Another solution is to use an air-lift system, whereby themedium is sparged with air from the bottom of the vessel (Toivonen et aL, 1990). Toivonen et al.demonstrated that there is no difference in the chemical profiles between shaker flasks and thefennenter.Various culture systems, including fei inenters, were examined. The chemistry of tissuecultured roots often reflects that of the roots. Examination of the roots of Ambrosia chamissonis(Chapter Two) indicated that the chemistry of the root varies depending on which part of the root isextracted. In this chapter the chemistry of normal root cultures was compared with that of hairyroot cultures.55Material and MethodsPlant MaterialMost collections of Ambrosia chamissonis were obtained from Centennial Beach,Tsawwassen, British Columbia. Samples were also taken from North Beach, Queen CharlotteIslands, British Columbia.Bacterial CulturesStrains A4, 15834, and TR7 of Agrobacterium rhiz,ogenes were obtained from Dr. L.Moore, Department of Plant Pathology, Oregon State University, Corvalis, Oregon. Bacterialcultures were grown up on Potato Dextrose Agar (Difco), stored at 25°C and then transferred toPotato Dextrose Broth (Difco) and placed on an orbital shaker at 100 rpm incubated at 28°C.Bacterial liquid cultures were used to prepare stocks which were stored in 50% glycerol at -70°C inEppendorf tubes.&plant PreparationsStems leaves and petioles were examined for their tissue culture potential. Roots wereexcluded because they are difficult to sterilize.Green stems which had widely spaced leaves were selected. They were defoliated,washed, and cut into segments 6-7 cm long. Under sterile conditions the segments wereimmersed, for 15 minutes, in a 30% bleach (Javex) solution with 2% Tween-20 (BDH) (Figure5.2). They were rinsed several times with sterile distilled water, washed in 70% ethanol for 2minutes and blotted dry on sterilized filter paper. The bleach-damaged ends were excised anddiscarded. The rest of the segment was cut into pieces 3-5 mm in length. Both normal andtransgenic mot cultures were generated from these segments.56Figure 5.2 Transformation of Ambrosia chamissonis Stem Segments with Agrobacteriumrhizogenes.Normal Root CulturesThe explants were placed on solid Schenck and Hildebrandt medium (Schenck andHildebrand 1972) and Murishige and Skoog medium (Murishige and Skoog) supplemented with57various concentrations of the auxins NAA (naphthalene acetic acid) and IBA (indole butyric acid)as well as the cytokinin BAP (benzylaminopurine)Transgenic Root CulturesThe explants once cut were immediately immersed into a 18 hour bacterial culture for 10minutes (Figure 5.2). They were blotted dry on sterilized filter paper and placed on plates ofwater/agar. Control segments were immersed in PD broth. After incubation for three days theywere transferred onto antibiotic SH plates. Carbenicillin (300 mg/L) and vancomycin (250 mg/L)were filter-sterilized through 22 pm millipore filters into medium that had cooled to less than 30°C.Preparation of AcetosyringoneAcetosyringone (7.84 mg) was dissolved in 100 ill DMSO and brought to 200 ml withbuffer (0.9% NaC1, MES pH 5.5) to make a 200 iaM solution. Various concentrations (0-200p.M) of acetosyringone were filter sterilized into the bacterial culture prior to treatment to see iftransformation was enhanced.Verification of TransformationTo verify that the hairy root cultures were transformed paper electrophoresis was employedto detect the opines (Petita al., 1983). Fresh root samples were ground in 1% HCI (1 mg/gtissue) for 10 minutes in a boiling water bath. The homogenate was centrifuged at 2000 g for 10minutes. The supernatant was evaporated under reduced pressure at 40°C and dissolved in water(0.2 mug tissue). Ten micmlitres of the extract was spotted on Whatman 3 MM paper andelectrophoresced at 90 V/cm (900 or 1500 V) for 8-11 min on a Desaga apparatus with a BioRadpowerpack. The buffer used was formic acid:acetic acid:water (30:60:910). The electrophorogramwas dried and dipped in silver nitrate solution prepared as outlined by Trevelyan a al., (1950).The papers were dried and then developed, at room temperature, in a solution of 20 g NaOH in100 ml H20 made to 1L with 95% ethanol (Ellis et al., 1984), immersed in fixer and washed for 358hours with running water. Standards of agropine, agropinic acid, mannopinic acid and mannopinewere kindly supplied by Dr. Petit (Groupe de Recherche sur les Interactions entre Plantes etMicroorganisms, Universite de Paris-Sud, Institut de Microbiologie, Orsay, France). Resultswere recorded as a positive or negative for the presence or absence of standard opines.A Southern blot was prepared with DNA from transformed roots with different strains ofA. rhizogenes and non-transformed roots, but the probe of T-DNA from A. tumefaciens used wasnot successful.Thiarubrine LocalizationHand sections of normal and hairy roots were made. Inititially JB-4 embedding techniqueswere employed, but due to the loss of thiarubrines and the success of hand sections they were notincorporated into this study. All sectioning was done in dim light to minimize the conversion ofthiarubrine to thiophene prior to viewing.Growth Curve of Transformed and Normal Root Cultures.Forty milliliters of SH medium were placed in 125 ml Erlenmeyer flasks and inoculatedwith 5 root tips approximately 1 cm long from normal root cultures or hairy root cultures generatedfrom Agrobacterium rhizogenes strain A4. The medium for the normal root cultures wassupplemented with 2 mg/1 of NAA. There was a total of 36 flasks set up for each line. Thecultures were maintained on a rotary shaker at 80 r.p.m.s in the dark at 24°C. Four flasks of eachwere weighed and analyzed every four days. Thiarubrine content was determined by UV-Visspectrometry. The four samples were combined and prepared for HPLC analysis.Chemical Analysis of all CulturesThe root cultures were blotted dry and then rinsed three times with distilled water andfrozen to -70°C. They were freeze-dried on a Virtis Lyophilizer. The dried tissue was weighed,ground and extracted with methanotacetonitrile (7:1) for 24 hours. The sample was filtered59through Whatman 1 filter paper. Overall thiarubrine content of the filtrate was calculated at thispoint using a molar absorptivity of 3000. The solution was evaporated under reduced pressure at28°C and resuspended in HPLC grade CH3CN. Filtering through glass wool was required toremove precipitates of more polar components. The filtrate was reduced in vacuo and resuspendedin HPLC grade acetonitrile. This was repeated until no residue was left on the side of the flask.The sample was passed through a Sep-Pak and injected into a Waters HPLC equipped with aphotodiode array detector. The column used was a Varian Micro Pak MCH-10 reverse-phasecolumn at 1 mllmin. The first runs were isocratic with CH3CN:H20 (72:28). Samples were theninjected and run isocratically with CH3CN:H20 (50:50).FermenterEight liter air-lift fermenters (manufactured by Dr. M. Hjortso, Dept. of ChemicalEngineering, Louisiana State University, U. S. A.) contained 4 liters of SH medium. Themedium for the normal roots was supplemented with 2 mg/L NAA (naphthalene acetic acid)whereas the transgenic roots (strain A4) were maintained in hormone-free medium. Eachfennenter was innoculated with two grams of roots. Air was supplied from the bottom and thefermenters were kept in the dark. After the cultures had reached stationary phase the media as wellas the roots were extracted and analysed for thiarubrines and thiophenes. The medium waspartitioned with chloroform, dried with MgSO4, concentrated in vacuo, and resuspended in HPLCgrade acetonitrile. Thiarubrine concentration was calculated. Two fennenters were set up for eachline.SulphateA low yielding transgenic mot line was chosen for this experiment to see ifsupplementation of sulphate would enhance the production. The control flask contained the normalSH medium that had been used for growth of the cultures. In treatment 1 the flasks were enhancedwith 0.8 g/L of MgSO4, in treatment 2 they were enhanced with 3.2 g/L Four flasks were set up in60each treatment and cultured in the dark at 24°C for 28 days. The cultures were extracted aspreviously described and the thiarubrine content was calculated per flask. Each sample wasanalysed by HPLC.Malic AcidSH medium was prepared with different concentrations of malic acid (0.1, 5.0, 10.0, 50.0,100.0 mg/L) and the pH adjusted to 5.6 with KOH. Four 250 ml Erlenmeyer flasks were set upfor each treatment Each flask contained forty milliliters of the medium and were innoculated withfour, approximately 1 cm long, tips of hairy roots transformed with A. rhizogenes 15834. Theywere incubated in the dark on a rotary shaker at 100 r.p.m.s at 24°C. The cultures were harvestedafter 28 days, blotted dry, freeze-dried and prepared for HPLC as already described.Cultures Initiated From Plants Collected From the Queen Charlotte IslandsPlants were collected from North Beach, Queen Charlotte Islands (see Chapter Two). Thetwo types were prepared for hairy root culture as already outlined. Only stem segments from type2 initiated roots. All of Type 1 stem segments were contaminated with fungi and could not beretrieved.RESULTSNormal Root CulturesCultures were found to grow best on SH medium with 4 mg/L NAA and the explant ofchoice was found to be young green stems. MS medium enhanced the production of anthocyaninpigments. These were detected by extracting with acidified methanol and examining the UV-Visible spectrum. The acetylenes were not stable in this extraction solvent. Although thethiarubrines were produced the cultures did not grow as rapidly as on SH, therefore SH mediumwas used for the rest of the studies. Over time the requirement for growth regulators changed and61the medium was subsequently altered with NAA and BAP to improve growth. Root cultureswhich did not exhibit callus growth were extracted.Hairy Roots Cultures and Opine DetectionAll strains of Agrobacterium rhizogenes employed, induced roots on the explants withintwo weeks of inoculation. Detection of opines using paper electrophoresis verified that the cultureswere transformed. Agropinic acid was detected in all hairy root cultures. Agnapine was detectedfrom hairy roots derived from A4 and 15834, whereas TR7 strains produced mannopinic acid andmannopine. In one case a line from TR7 opines were not detected. No opines were detected innormal root cultures.Transformation was apparent because the roots exhibited ageotropic growth characteristicof "hairy roots". The roots transformed with A4 and 15834 exhibited more extensive branchingthan those transformed with TR7. Transformation was efficient (3-8 roots initiated per stemsegment), therefore acetosyringone was not required.Chemistry of Root CulturesThe thiarubrine profile of the hairy root cultures did not differ from that of most normalroot cultures. Some of the latter possessed callus, therefore, the overall thiarubrines produced wasless than cultures comprised solely of roots. The data presented in Table 5.1 and Figure 5.3 wereobtained with normal cultures which did not exhibit callus formation. The problem with thenormal cell cultures was to keep the growth medium at the optimum hormone levels otherwisegrowth rate decreased and callus content increased.Cultures on medium supplemeted with BAP generated leafy shoots which ultimatelygenerated roots. Interestingly canals were evident in the leaves prior to formation of the roots.62FA 1aM 2aM 3a• 4 aO 7a/8aTable 5.1 Overall Thiarubrine Content of the Root Cultures.Root Culture Overall Thiarubrine Content(mg/g dry wt)Normal Root Culture 1.02 (± 0.40, n=7)Hairy Root-A4 1.24 (± 0.15, n=6)Hairy Root-15834 1.03 (± 0.50, n=9)Hairy Root-TR7 1.07 (± 0.24, n=7)Error is presented as standard deviation (n varies)63100 9 0 -8 07 0 -6 05 0 -4 0 -3 0 -2 0 -1 0 -0  NR^HR-A4^HR-15834 HR-TR7^Root-S^Root-WTissue Cultures and RootsFigure 5 . 3 Comparison of Thiarubrine Composition in Roots and Root Cultures."normal"=root cultures generated with growth regulators; 11R4lairy root cultures (different strains of Agrobacteriumare identified); Root-S=Succulent Root; Root-W=Woody RootLocalizationExamination of cross-sections of root cultures revealed that both transformed and non-transformed roots possessed easily discernible canals between the double endodennal layers.These red canals run longitudinally throughout the tissues. Root cultures exposed to light for anhour exhibited purple coloration. Upon sectioning the purple color was found within theendodennal cells themselves. These are presumably anthocyanins which may absorb light andprevent autotoxicity.64Figure 5.4 Cross-Section, Stained with Toluidine Blue, of a Transgenic Root.(t = thiarubrine canal)Figure 5.5 Cross-Section, Stained with Toluidine Blue, of a Transgenic Root. (t thiarubrinecanal, e = endoderm, pp = primary phloem, px = primary xylem)Growth curveThe growth curve of the root cultures was similar to that of other root cultures reported.There is a characteristic lag phase followed by a growth phase (logarithmic phase) and terminating0 . 5OD0 . 43r.a0 . 30 . 2T.tad 0 .10 -0 -0-0 -0-El Hairy Roots• Normal Roots1.1.1.1•1`1'4^8^12 16 2 0 24 28 32Day0.000^ 0.60:0.55-0.50:0.45:0.400.350.30:0.25:0.20:^ 0.00in a stationary phase. Although this pattern was observed in the transgenic and non-transgeniccultures stationary phase was reached much sooner in transgenic cultures (Figure 5.6).Figure 5.6 Growth Curves of Transgenic Root Cultures and Normal Root Cultures.650^4^8 12 16 20 24 28 32DayFigure 5.7 Growth Curve of Transgenic Root Cultures and Thiarubrine Production(in mg/ g dry wt.).300^350^400^450^500Wave ..ngth 200 ----00 n•The chemistry of the hairy root cultures changed over time. During the lag phase (to day12) a thiarubrine was observed at 52 minutes with H20:CH3CN (50:50) in higher quantities thanthe alcohol thiarubrines (Figure 5.8). This was intermediate between the thiarubrine 3a andthiophene 3 b. However, the small amounts precluded analysis at this time.Figure 5.8 HPLC Trace of 8 Day Old Hairy Root Culture Extract at a Flow Rate of 1 ml/min at50:50 (H20:CH3CN) with the UV-Vis Spectrum of an Unknown Thiarubrine (i).FomenterThe growth rate of the transgenic roots was more rapid than that of the non-transgenicroots. The hairy root cultures were ready to harvest at four weeks whereas the normal rootsrequired six weeks. In both cases the roots grew as a dense mat without callus formation. Themore polar thiarubrines were detected in the medium (Table 5.2). This is due to the branch rootswhich originate from the pericycle of the root inside the endodermis and rupture the cells of the66parent root releasing the contents of the canals. The chemical profiles of the roots (Figure 5.9) didnot differ from the shaker flask cultures. The increase in biomass was substantially enhanced inthe fermenter. In four weeks the transformed cultures grew from 1.0 g to 550.0 g. The weightwas not accurate because of the residual medium.Table 5.2 Thiarubrine Yield From Freeze-Dried Cultures from Fermenter.Tissue Extracted Overall ThiarubrineProductionHairy Roots 0.92 mg/g. dry wt.Normal Cultured Roots 1.12 mg/g. dry wt.Medium from Hairy Root Culture 0.050 mg/LMedium from Normal Root Culture 0.067 mg,/LHairy Root^Normal Root^Medium-HR^Medium-NRExtractFigure 5.9 Thiarubrine Content of the Cultured Roots (HR=Hairy roots transformed withAgrobacterium rhizogenes A4, NR= Normal Root cultures) and the Culture Medium.HPLC analysis of the medium revealed minute quantities of an unknown polyyne,suggested from its UV-Vis spectrum67SulphateThere was no significant difference between the dry weight of the samples or thethiarubrine content between treatments (Table 5.3).Table 5.3 Thiarubrine Production in Hairy Root Cultures (A4) Treated with Sulphate.Treatment (mg/L MgSO4)Dry wt. of rootsin gramsThiarubrine Contentin mg/g dry wt.0 (SH) 0.3977 (± 0.0212) 0.4689 (±0.1655)1 (SH + 0.8 g/L) 0.4135 (± 0.0480) 0.4720 (± 0.1510)2 (SH + 3.2 g/L) 0.3513 (± 0.0760) 0.3966 (± 0.0482)Mahe AcidThe cultures which were treated with malic acid were darker brown with increasingconcentration. Upon microscopic inspection one could see that the cortex of the roots wasproliferating while the endodermis, with its canals, remained intact. The concentration ofthiaurbrines decreased with increased concentration of malic acid (Table 5.4). HPLC profilesindicated that there was no difference chemically between the treatments except that there was onlytrace amounts of the thiarubrines and thiophenes of the primary alcohols, and the amount ofthiosulphinate 9 was enhanced (Figure 5.10) All of the cultures including the controls exhibitedpeaks which overlapped the peaks of the thiarubrines and thiophenes of the primary alcohols(Figure 5.11).68Table 5.4 Thiarubrine Production in Transgenic Cultures Treated with Various concentrations ofMalic Acid.Malic Acid Treatment(mg/L)Thiarubrine Concentration(mg/g dry wt)0.0 0.60 ± 0.03400.1 0.57 ±0.01415.0 0.53 ± 0.062110.0 0.49 ± 0.008350.0 0.38 ± 0.0484100.0 0.38 ± 0.0205Error is given as standard deviation, n=4, except in treatment 10, where n=3-_____.0-,....,.1.M2. ;.s.^..^iLiz_..,.:,....;,.^4b4a1,• L ^• •2104 >0^I • f t* 4' . !.370388450.^,..7b8b• •^'8a7a.TreatedUntreatedJ.^ 1. . ,1 0I^'^•^•20RetentionTime in Minutes'^I30I40Figure 5.10 HPLC Traces of Untreated Versus Cultures Treated with Malic Acid and the UV-Vis Spectrum of Thiosulphinate 9. Wavelengths are presented in nanometres.69,.^,_--^_______315 nm325 nm.^....^.-__•.2._.t.,._-...L__'4^•^•1^2tJ-:01^ 1^ 1^ 1^I^I10 20Retention lime in MinutesFigure 5.11 HPLC Trace of Extract from Malic Acid Experiment Representing Treated andUntreated Cultures with the "In flight" UV-Vis Spectra of Unknown Components.Transgenic cultures generated with Agrobacterhon rhizogenes (strain A4) from Type 2Queen Charlotte Island plants exhibited the same chemical pmfile as the other cultured roots,except that the concentration of thiarubrine 2a was higher than in the other cultures (30%).Subsequently thiarubrine la was lower in contration (64%), but the rest were approximately thesame: thiarubrine 3a (1%), thiarubirne 4a (3%), and thiarubirnes 7a and 8 a (2%). The overallyield of thiarubrines was found to be 1.15 (±0.25) mg/g dry wt.70DISCUSSIONTissue cultured roots, although a source of thiarubrines, did not yield large amounts.Enriching the growth medium with potential precursors, such as sulphate or malic acid, did notprove to be effective. This may be due to inaccessibility of sulphate or because the thiarubrines arebeing produced at their optimum concentration of this tissue system. Roots with secondary growthaccumulated these chemicals in canals in the developing peridenn. In the root cultures the canalswere limited to primary tissues which may have reached their physiological capacity. Although theChaenactis cultures in cross-section looked very similar to the Ambrosia root cultures thethiarubrine content of the former was twice that of the latter. Identification of the other constituentsof the canal may reveal the reason(s) for this.Since the TR7 strain of Agrobaaerium contains only one piece of T-DNA it was not theabsence of the genes encoding for the production of the opines which gave a nefative result in theopine detection procedure. Over time cultures may lose their ability to synthesize thesecompounds. The morphological characteristics of the culture and the fact that they grow on mediadevoid of growth regulators suggest that they are most probably transformed.Croes eta!. (1989) examined the role of auxin in root culture. Lateral branching of theroots is thought to be induced by high levels of endogenous auxin. Not only did the addition ofhigh concentrations of IAA cause the production of root primordia, but they indicated that theagropine-type strains of Agrobacterium cause more branching in transformed tissue. This is notsurprising since these strains introduce genes encoding for the production of IAA. Themannopine-type tested had much less in the way of secondary branching. The A4 and 15834strains both exhibited more extensive branching than TR7, however, there was no difference in theoverall production of thiarubrines. Croes et al. (1989) found that thiophenes accumulated in theroot tips which had been severed indicating that auxin is important for the synthesis of thiophenesbecause this is where it accumulated. Theoretically roots which have lots of lateral branchingshould produce more of these compounds. Croes eta!. (1988) indicated that the polyyne content71was highest in the roots tips. This does not appear to be true in light of the fact that the thiarubrinecontent of hairy roots transformed with TR7, a strain which does not cause the production ofauxins and consequently does not have as extensive lateral branching, does not exhibit lowerthiarubrine content that those transformed with strains which do encode for auxin synthesis.Gomez-Barrios et. al. (1991) examined the thiarubrine production in the hairy root culturesof Ambrosia artemisifolia. Not only did they use the cultures to gain the thiarubrines completelyassign 13C NMR data they also verified the biosynthetic pathway suggested by Bohlmann (1973),which stated that C13 dithiacyclohexadienes are derived from C14 precursors.Ambrosia chamissonis produces a wider array of the thiarubrines and therefore would bean interesting system to examine the biosynthesis of these compounds as well as to examine theinter-relationships between them.One area of phytochemistry that is now being examined is the biotransformation ofchemicals into more useful compounds through the use of tissue cultures. Many classes ofchemical reactions are involved in the biotransformation of exogenous substrates by plant cellculture. These include: hydroxylation, oxido-reduction of ketones and aldehydes, reduction of thecarbon-carbon double bond, glycosylation and hydrolysis. These processes are outlined in areview article by Suga and Hirata (1990). Suspension cultures have been traditionally the culturesystem of choice. These immobilized cultures have many advantages. Not only is sampling theculture over time easy, but addition of precursor and contact with virtually all cells in the system isaccomplished. Suspension cultures of Coffea arabica, Datura innoxia, Eucalyptus perriniana, andNicotiana tabacum fed (RS)-tropic acid, produced a number of glucosides and glucose-esters(Ushiyama and Furuya, 1988). Root cultures of Panax ginseng, using 2-phenolpropionic acid,produce glucose esters with high efficiency. Half of the product is excreted into the mediummaking this an ideal system for collection. Kawaguchi et al. (1990) demonstrated this by feedingPanax ginseng hairy root cultures digitoxigenin. Three esters and two glycosides were produced.In many cases glycosylation is desirable because it increases solubility of the product. Data are notyet available to compare the chemical output between suspension or callus culture systems and root72culture systems. It has become apparent that both systems have their merits. It may be that thesystems will modify chemicals in different ways. In short, plant cultured cells have a highglycosylation activity for compounds with a small Mr and a small number of substituents. The useof tissue culture may allow one to produce difficult to synthesize chemicals.Kennedy eta]. (1993) compared the chemistry of roots with transgenic root cultures ofArtemisia absinthium. This is not a reasonable comparison to make since the cultured roots areyoung and do not have secondary growth. A comparison should have been made betweentransgenic cultures and non-transformed root cultures. An interesting point which they did notaddress directly is the potential of root cultures as a system for determining where the componentscome from. In the plant resin canals often run through the roots and into the stem. The oilcomponents may have different sites of synthesis. Culturing shoots and shoots separately maygive some indication where the chemicals originate. Cultured roots are immature compared withthe roots from normal plants. Esau (1977) points out that, in some members of the Asteraceae,cells of the endodermis may divide tangential y resulting in a double endodermis and that secretorycanals can develop between the two-layers. This appears to be the case with Ambrosia culturedroots. However, more extensive investigation still needs to be done to clarify this.73CHAPTER SIXELICITATION WITH FUNGAL CELL WALL PREPARATIONS IN HAIRY ROOTCULTURES OF AMBROSIA CHAMISSONISINTRODUCTIONSecondary compounds are generally considered defensive in nature; the stresses of theenvironment can elicit their production. These stresses may induce the biosynthesis of novelchemicals (phytoalexins) or enhance the production of secondary metabolites already present inplant tissue. The phenomenon is known as elicitation and there are two major types of elicitortreatments: with biotic elicitors, which include preparations from pathogenic organisms such asbacteria and fungi, and abiotic treatments, which include the use of salts of heavy metals.Tissue culture is a good system for studying elicitation of secondary metabolite production.Suspension cultures have been studied the most extensively. Lithospermum erythrorhizonsuspension cultures, treated with a Penicillium preparation isolated from a contaminated culture,exhibited higher than normal levels of shikonin The elicitor was prepared by grinding thePenicillium , autoclaving it, and then adding the product to the suspension culture. Elicitedcultures which were extracted in situ had the highest levels of shikonin (Kim and Chang, 1990).Van der Hellen eta!. (1988) examined Tabemaemontana species, which produce the indolealkaloid, apparcine, in suspension culture. Upon elicitation a number of new compounds wereproduced which exhibited antimicrobial activity, and thus, presumably function as defensivechemicals in the plant Apparicine, the original compound of interest, was produced in lowconcentrations. This demonstrates the unpredictability of the natural products which will beproduced upon elicitation.The first consideration is the selection of a suitable elicitor preparation. In earlier worknatural pathogens were used to elicite production of chemicals. It appears that effective candidates74are not necessarily restricted to these species. Conversely, not all elicitors will work on allsystems. Schumaker et al. (1987), examining alkaloid production in Escholtzia, found the cellcultures would respond to elicitors derived from Penicillium and Saccharomyces, but not fromPhytophthora megasperma. Munkandan and Hjortso (1990), on the other hand, examinedthiophene production of Tagetes patula hairy root cultures treated with various fungal elicitors andfound all species, including those not pathogenic in nature, enhanced the production of secondarymetabolites. The second important consideration is the mode of application of the micro-organismto the culture system. Coculture has been employed, but since in most cultures, there is little if anyphysical defense against invading factors, the pathogenic species tend to overgrow the explant Apopular application utilizes complex preparations from the organism. The active components ofmany are unknown. Cell wall preparations are commonly performed by liberating the cell wallcomponents through autoclaving, thus ruling out the role of enzymes. Whether the activecomponents are naturally active or whether they are activated through heat is also not known.Treatment with cellulase and pectinase (derived from fungi) have also been shown to be activeelicitors (van der Heijden et al., 1988).Elicitation of differentiated tissue, such as root and hairy root cultures, have only recentlybeing examined. Fungal elicitors prepared from Phytophthora and Pythium have been shown toenhance the production of polyynes such as trideca-1,3,11-ene-5,7,9-yne in hairy root cultures ofBidens sulphureus (Flores eta!., 1988). Other species such as Carthamus tinctorius, Bidenspilosus and Tagetes also exhibited enhanced levels of polyynes after application of elicitator.Mukandan and Hjortso (1990) examined hairy root cultures of Tagetes patula and elicitedthiophenes with preparations of Fusarium conglutinans. Thiophene formation varied although allwere enhanced at elicitor treatments between 0.1 and 0.2 mg of glucose equivalents per ml ofmedium, especially bithienylbutinene (BBT). The most effective treatment was incubation with theelicitor for 48 hours.Abiotic systems have also proved fruitful. Robbins eta!. (1987) examined the productionof the isoflavans, vestitol and sativan in HR cultures of Lotus corniculatus elicited with7 5glutathione. The activity of phenylalanine ammonia lyase (PAL) was enhanced at least three-foldover the elicitation period. Vestitol production was enhanced whereas, sativan was produced atlow levels. The isoflavans accumulated in the medium as well as in the tissues. Robbins et al.,(1987) examined the response of tissue, of different ages, to elicitation and found that responsesdiffered depending on age and on dosage of elicitor.Recently Davies et al., (1993) showed that a glycoprotein, isolated from a crudeVerticillium dahliae preparation, elicited two different responses. In cotton and soybean cellsuspension cultures, the protein component induced the production of phytoalexins, whereas thecarbohydrate component caused the production of peroxide. This is one example of componentsof elicitor preparations exhibiting different chemical responses by the same plant tissue. Howthese responses relate to the plant as a whole is not known. It may be that responses to thedifferent compounds are organ-specific.Although the potential for the use of elicitors has been demonstrated, in some cases they donot enhance the production of the major characteristic secondary metabolites of a plant species orthe compounds desired. For example, abiotic elicitors (copper and cadmium salts) increase theproduction of sesquiterpenoids rather than tropane alkaloids in Datum stramonium hairy rootcultures (Furze eta!., 1991). This aspect of the elicitation phenomenon has been insufficientlyexplored and may actually lead to the development of "new" desirable compounds.In this study, the elicitation of hairy root cultures of Ambrosia chamissonis was examined.Their superior growth to normal root cultures make them better candidates for the examination ofthe production of thiarubrine compounds. Phytophthora megaspetma, a soybean pathogen, hasbeen used successfully for the elicitation of thiophenes in various culture systems. Transgenic andnon-transgenic root cultures, although producing thiarubrines and thiophenes, do not yield highconcentrations of these potentially useful chemicals (Chapter Five). Elicitation offers a system toenhance these chemicals by presenting a "perceived" pathogen.76MATERIALS AND METHODSHairy Root culturesHairy root cultures were started from stem tissue inoculated with Agrobacterium rhizogenesstrain A4 and established on solid SH medium (Chapter 4). Four to five root tips approximately 1cm long were used to inoculate 40 ml of SH medium in each flask. Thirty-five 125 ml flasks wereset up for each trial. The cultures were maintained in the dark at 23°C on a rotary shaker (100rpm) for 26 days.Elicitation with Fusarium oxysporum (FO) ElicitorA preparation of elicitor from Fusarium was received as a gift from Dr. Carl Douglas.Four 25 day cultures were inoculated with 3m1 of FO elicitor. The controls were given steriledistilled water. The cultures were incubated for 24 hours and then frozen, freeze-dried, extracted,and quantified as already outlined in Chapter Five.Fungal CulturesPhytophthora megasperma. f. sp. glycinea. cultures were obtained from Dr. Carl Douglasat the University of British Columbia. The oomycete was grown on solid V8 medium composedof 200 ml clarified vegetable juice, 3 g CaCO3, 20 g agar, and 800 ml dH20. Once established,500 ml of liquid medium in 2 L culture flasks, were inoculated with pieces of agar. The liquidmedium was composed of 1.5% sucrose, 2 g/L asparagine, 0.2 g/L MgSO4 x 71120, 10 g/L CaC12x 21120, 1.04 g/L K2HPO4, 20 mg/L13-sitosterol, 3 g/L CaCO3, 0.1 mg/L FeSO4 x 71120, 0.1mg/L thiamine-HC1, 0.1 mg,/L ZnSO4 x 71120, 0.02 mg/L CuSO4 x 51120, 0.02 mg/L NaMo04 x21120, 0.02 mg/L MnC12 x H20 and the pH adjusted to 5.7 or 9.0. They were incubated at 23°Cand harvested after 4 months of growth.77Elicitor PreparationOne hundred grams fresh weight of mycelia were harvested and washed in a Buchnerfunnel on 37 gm nylon mesh filter, with distilled H20 until clear. The mycelium was rinsed with 1L of 0.5 M NaC1, resuspended in 200 ml and homogenized in two portions in a Sorvall Omnimixerthen filtered through 37 gm nylon mesh. The residue was resuspended in 500 ml Tris/EDTA (20jiM Tris/25 gM Na2EDTA; 12.11 g Tris/46.52 g Na2EDTA in 5 liters of dH20), homogenized,and then filtered. The filtrant was resuspended in 500 ml Tris/EDTA and stirred at 4°C for 18hours. The slurry was then filtered and washed several times filtering under suction on 37 gmnylon mesh. The washing agents were: 1 L Tris/EDTA, 1 L dH20, 1 L CHC13:CH3OH (1:1), and1 L acetone. The residue was air-dried for 18 hours. This process yielded 8.8 g of myceliumpreparation which was suspended in 880 ml dH20 and autoclaved for 4 hours. After cooling thesuspension was filtered through 37 gm nylon mesh and the filtrate was concentrated toapproximately 1% volume under reduced pressure at 40°C. The concentrate was dialyzed against4 L of dH20 for 24 hours then was filtered through Whatman GF/A filter paper and centrifuged at10,000 x g for 20 minutes. The supernatant was filtered through 0.45 pm membrane filters andfreeze-dried. The elicitor was quantified in glucose equivalents determined by anthrone-glucoseassay (Dische, 1962). This is a conventional way of expressing the amount of elicitor; it is notintended to imply that the active ingredients are carbohydrate in nature.ElicitationThe elicitor was filter-sterilized and different concentrations (0.0, 2.5, 12.5, 25.0, 125,500.0 and 1000.0 mg glucose equivalents) were added to the 26 day old cultures. There were fourflasks set up for each treatment. The controls received sterile distilled water of the same volume (2ml). The roots were harvested after 48 hours, rinsed with sterile distilled water, blotted dry andfrozen at -70°C.7 8ExtractionThe roots were freeze-dried, ground and extracted with CH3OH:CHCN (7:1) overnight.The samples were filtered and total thiarubrine content for each was determinedspectrophotometrically (refer to Chapter Two). The extracts for each treatment were combined andprepared for HPLC (refer to Chapter Two).RESULTSThe cultures elicited with Fusariwn exhibited no overall enhancement of thiarubrineproduction over the elicitor concentrations tested. In fact at 1000 mg glucose equivalents per flaskthe thiarubrine level was lower. Further studies using this elicitor were not pursued.TABLE 6.1 Overall Thiarubrine Production of the Hairy Root Cultures Elicited with PmgElicitor.TREATMENT(mg *slu F/flask)OVERALL THIARUBRINE CONTENT (mg/g dry wt)Trial 1 Trial 20.0 1.30 ± 0.07 0.92 ± 0.072.5 1.28 ± 0.06 0.90 ± 0.0812.5 1.23 ± 0.20 0.93 ± 0.0825.0 1.25 ± 0.05 1.01 ± 0.13125.0 1.22 ± 0.14 0.78 ± 0.42250.0 1.26 ± 0.08 0.88 ± 0.15500.0 1.22 ± 0.16 0.87 ± 0.25*glu E= glucose equivalents as determined by antlarone glucose assay (Dische, 1962).Each trial was an average of 4 flasks, except for Trial 1 treatment with 1000 mg glu E which was three flasks. Erroris expressed as Standard Deviation.79The overall thiarubrine yields did not vary substantially with the treatments of Phytophthoramegasperma (Table 6.1). They did however show marked change in the chemical profile of theroot cultures over the various treatments (Figures 6.1 and 6.2). Synthesis of thiarubrine 4a wasenhanced at what appeared to be the the expense of thiarubrine 1 a. The values for the thiarubrineswith intermediate polarity were not expressed because their values were obscured due to peakoverlap of another compound(s), as exhibited by the UV-Visible spectra.The concentration of thiarubrine 1 a decreased as the concentration of elicitor increasedThiarubrine 4a on the other hand increased. In trial 2 at 250 mg Glu E/flask the concentration of4a fell. Upon inspection of the HPLC traces it was evident that there was a high thiophene 4 bconcentration, implying that the lower values may have been due to degradation.In all treatments including the control, two compounds previously undetected, wereobserved by HPLC, with CH3CN:H20 (72:28) at 1 nil/min, at 8.63 and 11.34 minutes. TheirUV-Vis spectra are similar to the unknown polyyne mentioned in Chapter 1. The spectra are verysimlar to those polyynes which have three triple bonds and one double bond according toBohlmann (1973) (Appendix 111). It probably has a hydroxyl function because it has the samepolarity as the thiarubrine with the primary alcohol function. The production of the thiosulphinateis slightly elevated, whereas the concentration of the primary alcohols are lower with increasingconcentration of elicitor. The minor thiarubrines and thiophenes are in such low concentrationsthat although they were detectable, but not quantifiable.80 1008 06 04 02 0081o^2.5^12.5^25^125^250mg Glucose Equivalents per FlaskFigure 6.1 The Effect on Thiarubrine Profile of Hairy Root Cultures Elicited with VaryingConcentrations of Pmg Elicitor-Trial 1.1008060402000^2.5^12.5^25^125^250mg Glucose Equivalents per FlaskFigure 6.2 The Effect on Thiarubrine Profile of Hairy Root Cultures Elicited with VaryingConcentrations of Ping Elicitor-Trial 2DISCUSSIONThe thiarubrines appear to be biosynthetically related (Figure 2.13) eg. thiarubrine 4a issynthesized through thiarubrine 3 a from 1 a. Elicitation results in the apparent conversion ofbiologically active thiarubrine 1 a into a related molecule (thiarubrine 4a) which has a higher levelof activity (Chapter 6). Thiarubrine 4a is synthesized through thiarubrine 3a from 1 a. This is anovel aspect of the elicitation phenomenon,These results imply that the conversion process occurs in the thiarubrine canals themselvesbased on the fact that the amount of 1 a, already present in the canals, is reduced while 4a isincreased. Localization of the tissue response to an elicitor has not been examined. Whenapplying a preparation there is little known about absorption by the cultured cells and subsequenttransport to sites of chemical response. One benefit in using suspension cultures is thattheoretically all cells are in contact with the medium and thus have the potential to respond. In rootculture systems, however, the chemical production may be localized and therefore may rely on thetranslocation of the elicitor, or message, to the site. There has been very little investigation tocompare suspension culture and root culture systems. Both systems have their merits and it maybe that each produces a distinct array of compounds.Elicitation not only shows potential for the enhancement of desired chemicals in tissueculture, but it also offers an experimental system to study the biosynthetic pathways and themolecular basis for the production of chemicals. As already stated suspension culture systemshave been more extensively studied. Phaseolus vulgaris suspension cultures have been used tostudy the regulation of isoflavanoid biosynthesis (Dixon et al, 1989). Dangl et al. (1987)developed protoplasts, which through elicitation with fungal cell wall preparations and UV light,excreted coumarins and flavonoid glycoside phytoalexins respectively, into the culture medium.They proposed this as a suitable system for analysing signal transduction and gene activation of thephenylpropanoid pathway from which these two classes of compounds biosynthetically arise. Inthis instance undifferentiated cultures have an advantage in their simplicity. For compounds, such82as the thiarubrines, which are particularly toxic to plant cells and must be sequestered into canals,differentiated tissues would be more suitable for studying biosynthesis.The new polyynes had not been detected previously. It may be that the was probablyinduced by the use of a different incubator in this experiment. They may be precursors to theacetylenic compounds.Although the potential of abiotic elicitors has not been explored in this study, they appear tobe useful as a system for the enhancement of desired chemicals. They may also shed some light onthe requirements of the plant in nature for synthesis of certain compounds. Corchete et al. (1991)demonstrated this when culturing Digitalis thapsi cells where he found that lithium enhanceddigoxin production. The plant grows in acid soil with a high concentration of lithium and theresults with cell cultures indicate that this metal plays an important role in the production of thiscompound. Ambrosia chamissonis, which grows in close proximity to the ocean, is exposed tomany ions, whose concentrations vary over time. Such ions may be a key to the enhancement ofthiarubrine production.83CHAPTER SEVENANTIBIOTIC AND ANTIFUlsIGAL ACTIVITY OF THE THIARUBRINESINTRODUCTIONThiarubrine 1 a, isolated from Chaenactis douglasii, displays antibacterial and antifungalactivity, whereas thiophene lb requires light for such activity (Towers et al. 1985). Thethiarubrine exhibits enhanced toxicity in the presence of UV-A light against numerous bacteria via acomplex mode of action (Constabel and Towers, 1989). It is not known whether it is thethiophene produced via photo-conversion or whether it is the process of conversion, which isresponsible. Irradiation of the thiarubrine shows reduced toxicity to yeasts (Towers et al., 1985).It was suggested that this is due to the light independent activity being greater than that of thephototoxicity of the corresponding thiophene. Towers eta!. (1993) examination of the biologicalactivities of other thiarubrines indicate that 3 a and 4 a are toxic at lower concentrations to the testorganisms. Cytotoxicity tests, using mouse mastocytoma cells, show that the toxicity of thesecompounds on mammalian cells is less than that of thiarubrine la making these better candidatesfor medicinal agents.There are different ways to assay for activity against micro-organisms. Disk-diffusionassays, also known as the Kirby-Bauer test, have been used extensively for the study of antibioticand antifimgal activities (Tortora eta!., 1989) and has been accepted by the Food and DrugAdministation (FDA) as a method of testing for antibiotic activity. An extract is deposited on apaper disk which is placed on a thin lawn of the test organism. Activity is quantified by measuringthe zone of growth inhibition around the disk. The problem with this method is that a large amountof the test compound is required and the results tend to be more qualitative than quantitative, sincecompounds may either diffuse differently or have different stabilities on the growth media.8 4Another method, the broth dilution test, requires less test compound. 96-Well Microtitek plates areused. Small amounts of compound can be applied and serially diluted. The test organism isapplied and growth is detected by absorbance after a stated amount of time, allowing accuratemeasurements of activity to be made. This system has been applied to mammalian cytotoxicitytesting where the test cells are usually cancer cells. This allows one to compare the dosage of theantibiotic required to kill a pathogenic organism with the toxicity to host cells. Mosmann (1983)established a technique using a tetrazolium salt to quantify colorimetrically the survival ofmammalian cells and thus measure cytotoxicity. The cells are grown up and subjected to thetetrazolium salt which is cleaved by living cells. The fonnazan produced in the reactionprecipitates, but can be solubilized with iso-propanol. Changes in pH dictate the color of themedium which interferes with absorbance readings of the blue MIT formazan measurement Thisis remedied by converting the phenol red to yellow by acidification. Living cells will thereforecause the medium to turn yellow whereas the media in the wells with dead cells will be purple.The intensity of the yellow will reflect the number of living cells. Readings are taken on an Elisaplate reader using reference wavelength 600 nm.The potency of a compound is quantified as the LC50, which is defined as the lethalconcentration of a chemical required to kill 50% of the test organism per specified unit of time.Quantification is done by plotting the concentration of the test compound along the x axis and thepercentage mortality along the y axis on logarithmic probability paper, leaving space for, but notplotting the 0% and 100% mortality points (Litchfield and Wilcoxon, 1949). There should be atleast two points above 50% mortality and two points below 50% mortality. A best-fit line is drawnthrough the points therefore representing the expected mortalities over the concentration range ofthe chemical. The value obtained at 50% mortality is called the LC50.Differences in the activities of thiarubrines may be due to the difference in stability. It hasalready been demonstrated that the diol is much more stable in aqueous solution and storage over85time (Towers et aL, 1993). Differences in pH may also dictate differences in stability which maybe reflected in differing levels of biological activities.The antifungal activity of thiarubrines is of particular interest because of the very lowconcentrations required to treat human pathogens, such as Cartdida albicans and Aspergillusfumigatus (Towers et aL, 1993, 1985). Thiarubrine la is toxic, at very low concentration, tothese organisms in the dark as opposed to bacteria for which light is required (Towers et al., 1985)Much higher concentrations of compound was required for toxicity toward bacteria.Comparisons of bioactivity of the thiarubrines against numerous organisms includinggram-positive and gram-negative bacteria, a yeast and other fungi is presented. Most of theseorganisms are pathogenic to humans. The bacteria include Pseudomonas aeruginosa, anopportunistic pathogen causing infections which are difficult to treat in immuno-compromisedpatients. Staphylococcus aureus is of interest because it commonly causes infections particularly inthe skin. Escherichia coli, long considered non-pathogenic, may cause serious infections. Bacillussubtilis, is considered non-pathogenic, but another species Bacillus anthracis is the causative agentof anthrax. The yeast Candida albicans causes diseases of the lungs, mouth, intestine andreproductive system. Aspergillus fumigatus causes a disease known as aspergillosis. Thisopportunistic fungus is a very serious problem to people with compromised immune defensesystems. Trichophytonmentagrophytes and Microsponim gypseum, which are dennatophytes,cause ailments such as ringworm. Two imperfect fungi were also tested. These were Fusatiumtrincticum. and Verticillium lateritium, although considered plant pathogens, other related specieshave been implicated in mycotoxicoses. The activities of thiarubrines previously unpublished werecompared ie. the chlorohydrins, the primary alcohols, and thiarubrine 2a. Antibiotic assays arenot presented for the thiophenes.86MATERIALS AND METHODSOrganisms for Antifungal, Antibacterial and Cytotoxicity AssaysCultures of Candida albicans strain UBC #54 were obtained from the UBC culturecollection. Aspergillus fumigatus (DAOM 150786) was received from the Canadian Collection ofFungus Cultures, Center for Land and Biological Resource Research, Agriculture Canada, Ottawa.Pseudomonas aeruginosa was received from Dr. Hancock, Microbiology Department, Universityof British Columbia. Trichophyton mentagrophytes, Fusarium tricinctum, Verticillium laterifium,and Microsporum gypseum were supplied by Dr. Robert Bandoni , Botany Department,University of British Columbia. Staphylococcus aureus, Bacillus subtilis, and Escherichia colicultures were supplied by the Microbiology Department at the Univeristy of British Columbia.Mouse mastocytoma P815 cells (ATCC T1B64) were maintained in fetal calf serum.Chemical PreparationAll thiarubrines were purified by HPLC as outlined in Chapters Two and Four. They werequantified spectrophotometrically using the molar absorptivity for thiarubrine la (e=3000 at 490nm) and resuspended in acetonitrile at a concentration of 0.1 mg/m1 for antifungal and antibacterialtests and 1.0 mg/m1 for mammalian cytotoxicity tests. Acetonitrile was determined to be non-toxicat the low levels attained in the bioasays All manipulations of the thiarubrines were done in dimlight. a-Terthienyl was a gift from Zyta Abramowski (University of British Columbia),Gentamicin and Fungizone were purchased from Gibco, and Nystatin was supplied by Sigma.Comparison of Antibiotic Activity of Thiarubrines using Paper Disk AssayCultures for Paper Disk Assays: Overnight liquid cultures of Candida albicans,Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudornonas aeruginosa grown inMueller Hinton (MH) medium were applied by swab to MH plates, except in the case of C.87albicans which was applied to Yeast Nitrogen Base (YNB) (Difco) and Sabouraud Dextrose(Difco) plates. Cultures of Aspergillus fumigatus, Trichophyton mentagrophytes, Pusan=tricinctum, Verticillium lateritium and Microsporum gypseum were established on YNB medium.Conidia were harvested and suspended in MH broth (-106 conidia/ml) and applied by swab toYNB plates.Disk Assays: 1.0, 0.1, and 0.01 i.tg, of each test chemical and control antibiotics, wereapplied to sterilized paper disks (Schleicher and Schuell #740-E) and placed on the plates preparedwith bacteria and fungal conidia. Control antibiotics were chosen depending upon test organism.Gentamicin was used in anti-bacterial studies, whereas nystatin was used for anti-fungal. a-Terthienyl was used for both. Six impregnated disks were placed on each plate. The bacterialplates and C. albicans were done in duplicate. After the cultures were allowed to equilibrate for 1hour in a 37°C incubator one set of duplicate plates was placed under UV-A lights for 2 hours andthen returned to the incubator. This permitted the compounds to diffuse prior to light treatment.The other fungal plates were not subjected to light, but grown at 23°C for varying incubationtimes. Measurements of all disk assays was done by measuring the zone of inhibition around thepaper disk. All assays were done twice whereas for the C. albicans plates were done three times(except for the 1.0 lig, which were done once). Disk diffusion assays for activity against C.albicom on Sabouraud dextrose (SAB) and YNB media were compared. These assays were donethree times in the dark and under UV-A light as already described.Quantitative comparison of antibiotic activity of thiarubrines using microtiter plate assays.Organism preparation: A 24 hour culture of Candida albicans, grown at 37°C in YNBmedium was diluted to 25 x 104 cells/ml. Aspergillus fumigatus conidia were isolated from 48hour cultures grown at 37°C and suspended in YNB and MR broth to 5 x 105 condia/ml. A 24hour culture of Pseudomonas aeruginosa, grown at 37°C in MH broth, was diluted to 2.5 x104cell s/ml.88Broth dilution assay: Two hundred microlitres of MH medium were added to the first row(8 wells) of sterile microliter plate (Falcon 3072 Microtest llPivi Tissue Culture Plates) using aTitertek Multichannel Pipette. The second row was left empty, whereas 100111 were added to therest of the wells. The test compounds were diluted in MN medium to 1 lig/m1 for C. albicans andA. fumigatus assays and 2 jig/m1 for P. aeruginosa assays in MH broth. Two hundred microlitresof test compound solution were added to each well of row 2. Two wells were set up for eachcompound. A series of 22 two-fold dilutions, starting with row two of the test samples, wasaccomplished over two plates. No test compound was added to the last row of either plate. Thestarting concentration was 500 ng/ml for testing against C. albicans and A. fumigatus whereas thestarling concentration for P. aeruginosa was 1 jig/mi. One hundred microlitres of the conidiasuspension were added to all wells except for the first row. Therefore the first row of each platecontained only growth medium (negative control), whereas the last row of each plate was devoidof test compound but reflected the normal germination of the conidia (positive control). The plateswere incubated at 37°C in the dark.C. albicans : Duplicate plates were set up. One with medium of pH 5.5 and the other withpH 7. After 24 hours the optical density (0. D.) was determined with the Titertek Multiskan at 620nm. A cotton swab was used to take samples from the wells with the minimal inhibitoryconcentration for each compound and applied to YNB plates to determine if the compounds werefungistatic or fungicidal.A. fumigatus : The 0. D. at 620 nm of each solution in a test well was read 24 hours laterto determine the germination of the spores and the development of the mycelia. After 48 hours theplates were examined and growth was recorded as a positive or negative for each concentration oftest compound and the minimal inhibitory concentration was determined.P. aeruginosa: Duplicate plates were set up. One set of plates was equilibrated in theincubator for 20 minutes and then irradiated for 25 minutes with UV-A light Growth of the89bacteria was determined after 48 hours by examination of the plate and a rating of positive ornegative for growth.Cytotoxicity TestsP815 mastocytoma cells were prepared at 106 cells/m1 in fetal bovine serum. Fiftymicrolitres of fetal bovine serum were added to each well of a sterile microtiter plate (Falcon 3072Microtestllfm Tissue Culture Plate) using a Titertek Multichannel Pipette except the first andsecond rows of wells. One hundred microlitres were added to the first row of wells. The testcompounds were suspended in medium to lug /400 pi (preliminary trial) and 0.4 jig/400(subsequent trials). One hundred microlitres were added to the second row of wells. The testsamples, in 50 pl aliquots, were diluted in series from the second row onward except for the lastrow. Fifty microlitres of the cell suspension were added to each well from the second one onwardincluding the final row. The initial concentration of the test sample was 1.25 tig/1.11 (preliminarytrial) and 0.5 pg/m1 (subsequent trials). The final row was inoculated with cells, but devoid of testcompound (positive control). The plates were incubated for 22 hours at 37°C. 344,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) was dissolved in 5 mg /ml of PBSand filter sterilized. Ten microliters were added to each well and the plates were incubated at 37°Cor 2 hours. One hundred microliters 0.04 N HC1 in isopropanol was added to each well and mixedthoroughly. The plates were read on a Titertek Multiskan at 620 nm.LC50 DeterminationThe absorbance gleaned from the Titertek Multislcan is used to calculate the % survivalwhich can be extrapolated to get % mortality values. Quantification is done by plotting theconcentration of the test compound along the x axis and percentage mortality along the y axis onlogarithmic probability paper. A straight line was drawn through the points therefore representing90the expected mortalities over the concentration range of the chemical. The LC50 value is takenwhere the line intersects 50% mortality.All bioassays were repeated at least twice. All manipulations of the test compounds wereperformed in dim light.RESULTSPaper Disk AssaysPseudomonas aeruginosa was not susceptible to the compounds at the concentrations testedusing the disk-diffusion assay, therefore the data are not presented. The susceptibility of the otherbacteria and C. albicans is presented in Table 7.1. The bacteria tested exhibited susceptibility whenirradiated with UV-A light, the gram-positive bacteria (B. subtilis and S. aureus) having the largestclearing zones. In the dark, E. coli was very resistant to all thiarubrines except thiarubrine 3 a.This activity was enhanced slightly by light. The other thiarubrines exerted activity only aftertreatment with UV-A light. Thiarubrines 1 a and 2 a were toxic at lower concentrations than theother thiarubrines to B. subtilis.On YNB medium at lower concentrations (0.01 jig/disk) the less polar thiarubines 1 a and2a exhibited no activity against Candida albicans in the dark. The other thiarubrines demonstratedactivity at this concentration having similar activity ie. clearing zones were approximately the samesize. At higher concentrations (0.1 jig/disk and 1.0 jig/disk) all the thiarubrines were active. OnYNB plates the activities, for all concentrations tested, of thiarubrines I a and 2 a were enhancedwhen irradiated, whereas the activities were reduced for the other thiarubrines. Thiarubrines 1 aand 2 a exhibited larger clearing zones. The initial results using 'YNB medium (Table 7.1)contradicted those of Towers et al. (1985) who observe less activity of thiarubrine 1 a whenirradiated. Therefore Sabouraud (SAB) medium, which they used, was tested (Table 7.2). Therewas reduced activity of thiarubrines 1 a and 2a after irradiation with UV-A.91Table. 7.1 Disk-Diffusion Assay to Determine the Relative Toxicities of Thiarubrines to Micro-organisms in Dark and 2 hr Exposure to UV-A Expressed as Zones of Growth Inhibition (mm).Thiarubrine(jig/disk)B. subults S. aureus E. coil C. albicansDark UV-A Dark UV-A Dark UV-A Dark UV-Ala 1.0 +1+ 25.0±0.0 +/+ 19.0±4.0 -/- 11.5±0.5 32* 35*0.1 1 -/- 12.5±1.5 -/- 11.5±1.5 -/- -/- 18.7±1.9 22.7±5.7as n.t. n.t. n.t. n.t. n.t. n.t. -/- 16.3±5.62a 1.0 +/+ 20.0±0.0 +/+ 14.5±1.5 -/- 9.5±0.5 31* 34* 0.1 - - 10.0±0.0 -/- 9.5±1.5 -I- -I- 17.3±2.5 23.7±3.30.01 n.t. n.t. n.t. n.t. n.t. n.t. -/- 15.0±4.13a 1.0 +/+ 8.5±1.5 8.0±0.0 9.5±0.5 8.5±0.5 9.0±0.0 29* 20*0.1 - - - - - - 7.5±0.5 -/- -/- 21.3±1.9 17.3±2.1 4a 1.0 +/+ 9.5±0.5 +/+ 9.0±0.0 -/- + - 30* 15* 0.1 - - - - - - + +......... -/- -/- 22.0±1.0 12.7±1.25a 1.0 +/+ 10.01-0.0 +/+ 10.0±2.0 - - +/+ n.t. n.t.0.1 - - -/- -/- 8.0* -/- -/- 16.0±3.7 12.3±0.9 6a 1.0 +/+ 10.0* +* 12.0* - - +* ^ 26* 19* 0.1 - - -/- -/- 9.0* - - * 20.0* 15*7a 1.0 +/+ 10.5±0.5 +/+ 10.5±0.5 -/- +/+ 25* 17*0.1 -/- -I- -/- 8.0±0.0 -I- -I- 18.0±1.6 13.3±1.7 0.01 n.t. n.t. n.t. n.t. n.t. n.t. 12.7±0.9 10.0±0.88a 1.0 +/+ 9.5±0.5 +* 9.5±0.5 4. +/- 30* 15* 0.1 -/- -I- -/- +/+ -I- -/- 21.0±1.0 15.0±0.00.01 n.t. n.t. n.t. n.t. n.t. n.t. 12.7±1.2 10.3±0.9Gent 1.0 13.5±1.1 11.5±1.1 13.5±1.5 16.5±0.5 11.0±2.0 12.0±0.0 8* (N) 8* (N)0.1 4- -I- -I- +I- - --I- -I- -I- (N) -I- (N)^4- -/- -I- 4- -I- -I- 4- (N) -/- (N)a-tart 1.0...._... - - 29.5±0.5 - 24.5±0.5 -/- -/- -* 20*0.1 -/- 15.0±1.0 - 15.5±0.5 -/- -/- -/- 10±0.5 0.01 n.t. n.t. n.t. n.t.-- n.t. -/- 9.0±1.0n.t.Values were averages of two bioassays except C albicans, which was tested four times, and those values with "*"which were tested only once. Standard deviations are presented."+" or "-" values represent activity which was detected but not measurable.(N) indicates the antibiotic tested was nystatin instead of gentamicinGent=gentamicin, a-tert=a-terthienyl, n.t.=-not tested92Table 7.2. Disk-Diffusion Assay to Determine Antimicrobial Activity of Thiarubrines Against C.albicans on on Different Media in Dark and 2 hr Exposure to UV-A, Expressed as Zones ofGrowth Inhibition (mm).Thiarubrine(114/disk)YNB Sabouraud (Difco) Sabouraud*Dark UV-A Dark UV-A Dark UV-Ala 30.3±1.7 35.3±2.7 33.5±3.5 31.5±0.5 32.3±1.4 29.8±1.62a 27.5±1.8 33.3±2.4 33.5±1.5 31.5±0.5 31.8±2.1 29.3±2.43a 23.5±1.5 18.5±2.1 23.5±1.5 18.0-1-0.0 23.7±1.7 17.0±2.54a 25.0±0.0 14.7±0.9 24.0±0.0 14.0±0.0 22.7±0.5 13.0±1.05a n.t. • n.t. 19.5±0.5 14.0±0.0 20.0±0.0 13.0±0.57a n.t. n.t. 20.0±0.0 14.5±0.5 20.0±0.0 13.5±0.5a-Terthienyl 44- 20.0±0.1 -/-/- 15.0±0.0 -/-/- 15.0±0.0Variation is given as standard deviation, n=3.Table 7.3 Disk-Diffusion Assay to Determine the Dark Toxicities of Thiarubrines to FungiExpressed as Zones of Growth Inhibition (mm).Thiarubrine(lig/disk)F. tricinctum V. lateritium M. gypseum 1: mentagrophytesla^0.1 15.0±0.0 8.8±2.5 25.0±5.0 >30/>300.01 -/- -/- -/- 14.0±1.00.1 15.0±0.0 8.8±2.5 22.5±7.5 >30/>30 ^0.01 -/- -/- -/- 12.0±3.00.1 15.0±0.0 12.0±0.0 27.5±2.5 >301>300.01 4- +/- -1- 23.5±1.50.1 20.0±0.0 15.5±0.5 25.0±5.0 >301>300.01 -/- +/- -/- 11.5±0.50.1 14.0±1.0 13.0±2.0 25.0±5.0 >301>300.01.,.... -/- +/- +/- 15.5±0.50.1 15.5±0.5 14.5±0.5 26.5±6.5 >301>300.01 -/- +/- +/- 16.5±1.50.1 17.5±1.5 12.5±0.5 25.0±5.0 >301>300.01 -/- +/- -/- 13.5±1.5Nystatin^0.1 15.0±0.0 12.0±0.0 28.0±2.0 >30/>300.01 8.0±0.0 -/- 12.0±2.5 14.4±0.5Incubation times are given in brackets. Variation is given as standard deviation, n=3.93In Table 7.2 the toxicity of the thiarubrines against C. albicans was compared on YNB andSAB media. The more polar 3 a and 4a did not respond differently on the two media whereas 1 aand 2a differed in their responses to light. On YNB, as indicated in Table 7.1, the activity of thesethiarubrines was enhanced upon irradiation. On SAB there was a reduction in activity like the restof the compounds tested. a-Terthienyl was active only in the light.The chemicals were toxic toward all of the fungi tested (Table 7.3). They had similaractivity to nystatin as indicated by the clearing zones. The compounds were especially activeagainst T mentagrophytes. The chemicals are tested on the same medium which meansdifferences in activity toward the different organisms tested, can be attributed to differential toxicitytoward these organisms.The cytotoxicity test on the P815 mastocytoma cells were initially performed at highconcentrations of test compounds to find out what the concentration range of the tests should be.Several trials were performed, but due to problems with cell growth, calculations of LC50 werepresented for one run for which cell growth was good. These data are presented in Table 7.4.When plotting the LC50 curves the slope of the line for thiarubrine 1 a was shallow compared to theothers. Thus, there is a bigger difference between the MIC and the LC50 for thiarubrine 1a ascompared with the others. Other thiarubrines, such as 4a, 5a and 6a have steeper slopes andtherefore the MIC and the LC50 are closer in value.Preliminary studies were performed to establish the duration of incubation and the pH ofthe medium for broth dilution test on C. albicans. The LC50 was evaluated at 24 hours and at 48hours for activity against C. albicans in YNB medium at pH 5.5 and pH 7.0. At 24 hours therewas a much lower LC50 for the compounds in pH 7.0 than 5.5 probably due to a longer lag periodin the growth at that pH. 48 hour incubation time was chosen as the optimum incubation timebecause it gave the best cell growth. These results are presented in Table 7.4. Two trials werecarried out and data from both are presented. The samples from the wells which did not exhibit94Table 7.4 Comparison of the LC50 and Minimum Inhibitory Concentrations (MIC) For P815and Candida albicans.IThiarubrineLC50 and MIC During 24 hr Exposure in ng,/m1P815 (24 hours) C. albicans (48 hour)LCso MIC LC50trial 1 / trial 2MICtrial 1 / trial 2la 8.0 500.0 50.0 / 50.0 500.0 / 250.02a 63.0 500.0 35.0 / 90.0 500.0 / 500.03a 37.0 250.0 7.0 / 16.0 250.0 / 62.54a 9.0 31.3 1.3 / 3.5 7.8 / 7.85a 70.0 250.0^. 0.5 / 4.0 15.6 / 15.66a 16.0 62.5 3.5 / 7.0 31.3 / 31.37a 60.0 250.0 0.8 / 7.8 7.8 / 31.38a 21.0 125.0 0.3 / 6.0 7.8 /15.6Fungizone - >500.0 35.0 / 37.0 160.0 / 160.0Table 7.5 Toxicity Indices of Thiarubrines for P815 and C. albi cans.ThiarubrineLC50P815 MIC P815LC50 C. albicans MIC C. albicansla 0.16 1.001.00 1.003a 3.22 1.604a 3.75 4.005a 31.10 16.036a 3.04 2.007a 13.95 12.798a 6.67 10.6895growth (MIC) when spread on nutrient medium, did not resume growth, indicating that the testcompounds are fungicidal rather than fungistatic. Toxicity indices calculated using the LC50 andMIC values are presented in Table 7.5. The values for P815 cells are divided by the C. albicansvalues.Table 7.6 Broth Dilution Assay to Determine Minimal Inhibitory Concentration (MIC) for (A)P. aeruginosa in the Dark vs. 2 hr Exposure to UV-A Light and (B) A. fumigatus for two DifferentMedia.ThiarubrineMIC During 48 hr Exposure in ng/ml(A)P. aeruginosa ,dark^'^UV-A(B)A. fumigatus YNB^I^M-Hla >500^: >500 250^j_^2502a >500^1..,i>500 500 2503a >500^1 >500 500 5004a >500^1, >500 125 2505a 500 >500 125^2506a 500 >500 125 500i7a >500^i >500 250  ^125/250*8 >500 ^I >500 125 250Gentamicin >500^1 >500 250 500tests were done twice. Differing results are presented*. A value of >500 means that there was no activity at1000 pg/ill, but there was at 500 pg.P. aeruginosa and A. fumigatus were not susceptible to the test compounds except at veryhigh concentrations (Figure 7.6). There was a slight enhancement of activity with light for P.aeluginosa. The A. fimigatus was tested on two different media. MH media is a basic mediumfor bacteria and was devloped for toxicity testing.96AllDISCUSSIONIn the disk-diffusion assays the thiarubrines did not exhibit toxicity toward P. aeruginosain either light or dark, whereas E. coli was susceptible only after exposure to UV-A light except inthe case of thiarubrine 3a which was also active in the dark. The gram-positive bacteria, S. aureusand B. subtilis, were susceptible in light, and to a lesser degree, in the dark. The differencesbetween gram-negative and gram-positive bacteria are based on the presence in the former of amore complex cell wall. Gram-negative bacteria are generally more resistant than gram-positive toantibiotics, which is not surprising since most antibiotics act on the bacterial cell wall. Towers etal. (1985) found that thiarubrine la was more toxic to the yeasts S. cerevisiae and C. albicans inthe dark. The present study reveals that light activity is dependent on the medium used whentesting. The larger clearing zones for thiarubrines la and 2a indicate that these compounds may bemore soluble in the growth medium. At lower concentrations, however, thiarubrines la and 2aexhibited no activity toward C. albicans, whereas the other thiarubrines were active in both lightand dark implying that the clearing zone is not a good indicator of potency.It has already been indicated that the medium is very important when testing chemicals.Some media nullify the effects of antibiotics. Hoeprich and Huston (1976) tested numerousantifungal compounds, using four types of media. SAB and YNB were found to exhibit differentMIC's depending on the compound tested. They found that the synthetic formulations (YNB andSynthetic amino acid medium) did not antagonize the activity of miconile or clotritnazole, whereasSAB and brain-heart infusion, which are undefined media, did. Hoepich and Finn (1972)suggested that the presence of pyrimidines and pyrimidine ribosides, in the medium, is at leastpartially responsible for this.The results for C. albicans, in the broth dilution assays, were presented in two trials ratherthan averaging because some of the values varied for some chemicals tested. Nonetheless it isevident that the more polar compounds are active at lower concentrations than the more non-polar971 a and 2 a. It must also be kept in mind that the compounds are tested in serial dilution whichwould have a larger difference in the test concentrations at the higher concentrations, whichaccounts for the large differences in these values. To gain more accurate MIC values forthianibrine 1 a, for example, one would have to set up tests for concentrations between 250 and500 mg/ml. The purpose of this paper is to emphasize the potential of the more polar ones.For a compound to be a potential drug the LCso and the MIC values for a cytotoxicity test,such as the P815 assay, should be higher than those for the pathogen (C. albicans) This means thatthe low levels required to kill the yeast will have little effect on the host cells. Thiarubrines 5 a, 7a,and 8 a are the best candidates form this point of view as demonstrated by the toxicity indicespresented in Table 7.5. Thiarubrine 5a, for instance, can be applied in a concentration 1/31 adosage which is lethal to 50% of the mammalian cells, whereas the dosage of thiarubrine larequired to kill 50% of the C. albicans is lethal to the mammalian cells. Both the MIC and the LCsocalculations are important as is demonstrated for thiarubrine 1 a, where the LCso was much lowerthan the MIC. There is toxicity over a wide range of concentration as opposed to a compound suchas thiarubrine 5a, in which the mortality curve, when calculating the LCso, was very steep,indicating there is a critical concentration where the compound is toxic and therefore the MIC andLCso are relatively close.The mechanisms of action of the thiarubrines are not known, although it has beensuggested that the disulphide ring is important to biological activity (Constabel and Towers,1989c). They exhibit activity in the light and in the dark. The light requiring toxicity is probablypartially due to the toxicity exerted by the thiophene molecule which is produced after exposure tolight. Some of the toxicity may result from the conversion process itself. Antimicrobialcompounds have numerous modes of action. Some drugs inhibit the synthesis of the cell wall (eg.vancomycin) whereas others cause injury to the plasma membrane (eg. nystatin). Protein andnucleic acid syntheses can be inhibited as well as enzyme activity.98There is evidence that phototoxicity of thiophenes is due to the production of singletoxygen and subsequent oxidation of lipid components of membranes (McRae et al., 1985). Therehas been some work to determine the mode of action of these compounds. Two well-studiedcompounds of acetylenic origin a-terthienyl and phenylheptatriyne (PHT) have been examined fortheir activity. It was originally believed that the thiophene was oxygen dependent in its activitywhereas PITT was active aerobically and anaerobically (Weir et al., 1984). Kagan eta!. (1984)found that upon irradiation, PHT was a good singlet oxygen producer. This activity was shown torequire oxygen. It may be that the procedures employed by the former group did not accomplishan anaerobic environment. Gong eta!. (1988) examined the effect of KIT on red blood cellsirradiated with near UV light. They found that again there was a dependence on oxygen fortoxicity. The membrane proteins were established as the targets.Constabel and Towers (1989c) examined the structural characteristics, of the thiarubrines,which contribute to the biological activity by comparing the activity of thiarubrine la with that ofthiarubrine 11 a. The difference in these molecules is the triple bond of the former is replaced by adouble bond in the latter. There was no difference in the light-independent activities. Theyconcluded that the disulphide ring is the most important feature for activity of the molecules. Themode of action of the dark reactions as well as the light reactions is not known. Thiarubrine la hasbeen shown to have light-requiring anti-viral activity (Hudson, et al., 1987). Membrane virusesare more susceptible indicating that the target may be the membrane.Useful antimicrobial drugs must possess a number of properties. The most importantbeing selective toxicity. The compound should be toxic to the pathogen and exhibit little or notoxicity toward the host. Because bacteria and eucaryotic cells differ so much in compositionbacteria are generally easier to treat selectively than eucaryotic cells. Fungi, on the other hand arevery similar to higher organisms at the cellular level and therefore may be more difficult to controlwithout negative effects to the host The second criterion is that the drug must be soluble in bodyfluid. This problem has slowed the development of the drug taxol for the treatment of cancers.99100Due to its low solubility a large quantity of carrier solution has been required for its applicationwhich is itself toxic. Thirdly, the drug must be stable in both the body and on the shelf. Theserepresent only a few of the many important features.It has been demonstrated that thiarubrines have great potential as antifungal andantibacterial agents. A fundamental problem with evaluating them, based on bioassay results, isthat the conditions may not reflect the physiological environment of application. It is veryimportant, when testing these compounds, to employ bioassays which are appropriate. Whenexamining chemicals for medicinal activity the experimental conditions should reflect those inwhich the drug would be administered, as well if one is studying the activity of these compoundsin nature it is best to mimic the environment of the chemicals.Tested independently these compounds demonstrate a high level of toxicity to a number oforganisms. It is important to keep in mind that in the natural system they co-occur not only witheach other, but also with other compounds. There is little doubt that these compounds have adefensive role; the question is how do they operates in the plant. Toxicity in nature often infersthat there is some potential against pathogens in other systems. There is an extensive amount ofscreening chemicals must go through before they will be accepted as medicinal agents. Initialscreenings on crude samples are important This is best accomplished by quick and easy tests suchas disk-diffusion testing. Fractionating must be done to determine what the active components are.CHAPTER EIGHTGENERAL SUMMARYIt is important to emphasize the potential of the plant root as a chemical factory for theproduction of medicinal compounds. Roots, for the most part, have been under-utilized as asource of interesting and useful chemicals and often they are important sources of novel drugs, eg.ginseng. In these times of concern for the conservation of our forests and plants in general, tissueculture has potential as a source of plant-derived pharmaceuticals. Unexpectedly, a novelthiarubrine and thiophene were isolated from roots and root cultures, making it possible to explorespectroscopic techniques to identify them. Ambrosia chamissonis produces a family of thesesulphur heterocyclic compounds with dramatic antifimgal and antibacterial activities. There is littleknown about the biosynthesis of these thiarubrines and thiophenes. Data with elicited culturessuggest that at least the conversion from one thiarubrine to another (la to 4 a) takes place in canals.It is possible the synthesis of the basic structure of thiarubrine 1 a also occurs here. Although thereare many modifications to 1 a, thiarubrine 2 a is present without related derivatives. Manymembers of the Asteraceae produce only thiophenes such as Echinops, which produce anextremely wide array of thiophenes based on the structure of thiophene 2 b, without the presence ofthe thiophene 1 a type.Canals containing the thiarubrines can be seen throughout the plant. Anatomical andchemical investigations showed the canals to contain an oil in which the active thiarubrine andthiophene occur. The oil appears to have a stabilizing effect on the these light-sensitivecompounds, as indicated by their presence in parts of the plants exposed to sunlight eg. leaves.Studies on the bioactivity against numerous pathogenic fungi and bacteria indicated thatthese compounds differ in their activities. Thiarubrine 1 a which was the first to be examined, insome cases was more potent against yeasts. It was demonstrated that because of their high toxicityto mammalian cells (presented as toxicity indices) it has little potential as a therapeutic agent,1 0 1102whereas thiarubrines 5 a and 7a are less toxic to mammalian cells at the doses required fortreatment of the pathogen. Thiaubrine 3 a displayed antibacterial activities that the otherthiarubrines did not exhibit. How these compounds interact with the test organisms remainsunknown. 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Cell, 56: 193-201.APPENDIX I: Thiarubrines and Their Corresponding Thiophenesla CH3—C-=-C^ CH=C H2S S3-(1-propyny1)-6-(5-hexen-3-yn-1-yny1)-1,2-dithiacyclohexa-3,5-diene (Thiarubrine A)lb^CH3 C)2 C H = C H22-(1-propyny1)-5-(5-hexen-3-yn-1-yny1)-thiophene2a^CH3— (C C)2 C=-=-C—CH= CH23-(pent-3-yn-1-yny1)-6-(3-buten-1-yny1)-1,2-dithiacyclohexa-3,5-diene (Thiarubrine B)2b CH3— (C CH22-(pent-3-yn-1-yny1)-5-(3-buten-1-yny1)-thiophene3a CH3—C C/0\C)2 — CH— C H23-(1-propyny1)-6-(5,6-epoxyhex-3-yn-1-yny0-1,2-dithiacyclohexa-3,5-diene (Thiaubrine D)3h CH—CC/0\C)2 C H C H21092-(1-propyny1)-5-(5,6-epoxyhex-3-yn-1-yny1)-thiophene4a CH3 C C C)2— CHOH— CH2OH11 03-(1-propyny1)-6-(5,6-dihydroxyhex-3-yn-l-ynyI)-1,2-dithiacyclohexa-3,5-diene (Thiarttbrine D)4b CH3 C C CHOH— CH2OH2-(1-propynyI)-5-(5,6-dihydroxyhex-3-yn-1-yny1)-thiophene5a^ C)2 — CHCI—CH2OHs—s3-(1-propyny1)-6-(5-chloro-6-hydroxyhex-3-yn-1-yny1)-1,2-dithiacyclohexa-3,5-diene5b CHCI—CH2OH2-(1-propyny1)-5-(5,6-dihydroxyhex-3-yn-1-yny1)-thiophene6a CH3—C C C)2— CHOH— CH2C13-(1-propyny1)-6-(6-chloro-5-hydroxyhex-3-yn-1-yny1)-1,2-dithiacyclohexa-3,5-diene6b CH3—C=-C^3_(c.c)2_ CHOH— CH2C I2-(1-propyny1)-5-(5-chloro-6-hydroxyhex-3-yn-1-yny1)-thiophene8a^CH3—C7=-- C if' C)2 — C H2— C H2OHS3-(1-propyny1)-6-(6-hydroxyhex-3-yn-l-yny1)-1,2-dithiacyciohexa-3,5-diene8b (c -7=r= c)2 — C H2 — C H2OHII^I7a HO — CH2—C C^(C^—S S3-(hydroxyprop-1-yny))-6-(5-hexen-3-yn-yny1)-1,2-dithiacyclohexa-3,5-diene7b^HO — C112—C-===-X (C C)2 — CH= C H22-(hydroxyprop-1-yny1)-5-(5-hexen-3-yn-yny1)-thiophene2-(1-propynyl)-5-(6-hydroxyhex-3-yn-1-yny1)-thiophene9^CH3—C-=-C^(C=.02— CH—CH,S—S3-(1-propyny1)-6-(5-hexen-3-yn-1-yny1)-cyclohexa-3,5-diene-1,2-thiosulphinate10aCH3—C=--=-C (  ^— CH—CH— CH—CH,S—S3-(1-propyny1)-6-(3,5-hexadien-l-ynyl)-1,2-dithiacyclohexa-3,5-diene 11210b — CH— CH— CH= CH22-(1-propyny1)-5-(3,5-hexadien-1-yny1)-thiophene0C C CH— CH2lla /S3-(pent-3-yn-1-yny1)-6-(3,4-epoxybut-1-yny1)-1,2-dithiacyclohexa-3,5-diene0/CH2lib CH3—(C1--,--C)22-(pent-3-yn-1-yny1)-5-(3,4-epoxybut-1-ynyI)-thiophene12a CH3—CH=CH C C CC— CH=CH2S3-(pent-3-en-1-ynyI)-6-(3-buten-1-yny1)-1,2-dithiacyclohexa-3,5-diene12b CH3—CH=CH C C CC CH=CH23-(pent-3-yn-1-yny1)-6-(3,4-epoxybut-1-yny1)-thiophene113APPENDIX II(b): UV-Visible Spectra of Thiarubrine 2 a and Thiophene 2 b114APPENDIX II(c): UV-Visible Spectra of Thiarubrine 3a and Thiophene 3 b.I 15Thiarubrine 4a1^ 1250^300 350^400^450^500^550^600Wavelength 200-600 nm2001^1'111^1^1^1'111APPENDIX II(d): UV-Visible Spectra of Thiarubrine 4a and Thiophene 4b.1 16200^250 300 350^400Wavelength 200-400 nmAPPENDIX H(e): UV-Visible Spectra of Thiarubrine 5a and Thiophene 5b.1^1^.^1^1^1^1^.^1^1^1^•^1^1^1,1^I^1^1^1^1 ^1^1^1^1^.^1^1^1^1^1^1^I^1^.^1^1^1^.^1 1^1 ^Thiarubrine 5aI^1^1^1^1^1^1^1^1^f^1200^250^300^350^400^450Wavelength 200-600 nmI^1^1^1^1500 550 600117^1^1'1'1'1'1^I^1^1^I^1^1200 250 300 350 400Wavelength 200-400 nmAPPENDIX II(t): UV-Visible Spectra of Thiarubrine 6a and Thiophene 6b.200^250^300^350^400Wavelength 200-400 nm118400200^250^300^350Wavelength 200-400 nm200^250^300^350^400^450Wavelength 200-600 nm,1.1,1,11,1.1.1^1.1^1,1,1,1,1,1.1.1Thiophene 7bSOO^550^BOOAPPENDIX II(g): UV-Visible Spectra of Thiarubrine 7 a and Thiophene 7b.1^1^1^1.1,1,1,1,1.1,1.1.1,1,1,1^1,1^1^1^1^1^1^1^1^1^1^1^1^1^.1,1^1^1. Thiarubrine 7a111111'111^1'1'1'1'1'1'1.1'1'1'1 19APPENDIX II(h): UV-Visible Spectra of Thiarubrine 8 a and Thiophene 8 b.1,1A1,1,1,1,1,1■1•1.1,1.1,1,1,1,1.1,I,1^ 1,1,1 Thiarubrine 8a200^250^300^350^400^450Wavelength 200-600 nm^1.1,1,1^1.1^I ^Jill^I^I^I^I^I^t^I^1.1'1.1'1'/'200^250 300 350^400Wavelength 200-400 nm500 550 600120APPENDIX II(i): UV-Visible Spectrum of Thiosulphinate 9.121APPENDIX III: UV-Vis Spectral Data for Unknown PolyynesUV-Vis MaximaUnknown Polyyne #1Unknown Polyyne #2Unknown Polyyne #3Known PolyyneLong wavelengths group358^336^316358^336^316350^328^308356^333^312Lower wavelengths group286^272286^272292^270^260283^271UV-Vis Spectra122APPENDIX IV: Mass Spectrum of Isolated Crystals (Chapter 2)123APPENDIX V: Sesquiterpene Lactones from Ambrosia chamisonis1124Chamissellin^ ChamissoninCostunolide^ChamissanthinChamissarin^LaurenobiolideAPPENDIX VI: Electron Impact High Resolution Mass Spectrometry of Thiarubrine 8 aC H S^0 DEV MEAS MASS #PTS %INT13 10 2 1 0.1 246.0174 35 56.2512 7 2 0 -0.4 214.9985 29 51.2913 10 1 1 0.9 214.0461 29 26.9012 7 1 0 -0.4 183.0264 29 41.5311 7 1 0 -0.7 171.0262 35 58.5511 7 0 0 -0.8 139.0539 29 32.72125APPENDIX VII: Spectral Data of Thiarubrine 8 a and Thiophenes 7 b and 8 b7 b: UV Amax nm 357, 342(sh), 334, 249. MS (of TMS derivative) m/z (rel.int): 284 [Mr -(58), 195 [M-OTMSr(62).8a: UV Amax nm 490, 345. MS (of TMS derivative) m/z (rel. int.):246[M] -(38), 214 [M-S] -(83), 215 [M-CH20H](83), 183 [MS-S-CH20H] (100).8b: UV Amax nm 341, 322, 246, 235. MS (of TMS derivative) m/z (rel. int.):286 [M] -(33), 183 [M-CH20'TMS](10), 103 [CH20TMSr(42).126


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