POTENTIAL FOR USING INSECTS TO GUIDE THE SEARCH FOR MEDICINALLY-ACTIVE CHEMICAL COMPOUNDS IN PLANTS
Ciara Raudsepp-Heame Department of Plant Science - NEO McGill University, Montreal Submitted July 2003
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Masters of Science
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Tropical insects are weIl known for their diversity of defensive secondary compounds, most ofwhich originate in the insect's host plant. These have similar structures to compounds that are responsible for most pharmacological activity in plants and that are mined by pharmaceutical companies for drug development. There is a potential for using bioprospecting projects in the biodiverse tropics as an economic incentive to preserve the forests that are the source of potentially lucrative and life-saving new medicines. This thesis investigates the possibility of using aposematic insects as guides to plants that contain pharmacologically-active compounds. Various plants that were found to have preliminary activity against cancer, leishmaniasis, malaria or Chagas' disease were investigated to determine whether they were associated with an aposematic insect. Active plants that were found to have an interesting ecological relationship with at least one herbivorous insect were subjected to bioassay-guided fractionation using HPLC and standard chromatographic techniques in order to isolate the active chemical component of the plant. Plants were monitored within national parks in the Republic of Panama over a period of six months and aIl insects feeding on them were collected and raised in captivity. The insects were then extracted and analyzed to œtermine how they were treating toxic chemical compounds in their host plant. Two principle plants were investigated with their associated insects: (1) Vismia baccifera and (2) Mikania guaco. One generalist and one specialist Lepidopteran species were found to sequester vismione B from their host plant Vismia baccifera, a cytotoxic compound active against three cancer celllines. Two specialist Coleopterans were found to sequester the novel compound Guacanone, isolated by the primary author from the vine Mikania guaco and active against Trypanosoma cruzi, the causative agent of Chagas' disease. A generalist Coleopteran was found to not sequester this compound. RÉSUMÉ
Les insectes tropicaux sont reconnus pour la diversité de leurs composés chimiques dont la plupart proviennent des plantes hôtes. Ces composés chimiques de défense ont des structures similaires aux composés chimiques dans les plantes qui sont exploités par l'industrie pharmaceutique pour la mise au point de nouveaux médicaments. Les projets de bioprospection dans les tropiques, riches en biodiversité, pourraient d'ailleurs inciter la préservation des forêts. Ces forêts abritent les sources de médicaments éventuellement lucratifs et profitables pour la médecine. Ce mémoire de maîtrise étudie la possibilité d'utiliser les insectes aposématiques comme indicateurs pour identifier les plantes qui contiennent des éléments pharmacologiques actifs. Plusieurs plantes sont connues pour leurs activités préliminaires contre le cancer, la leishmaniose, la malaria ou la maladie de Chagas. Celles-ci ont été étudiées afin de déterminer si elles sont liées à des insectes aposématiques. Les plantes actives qui ont une relation écologique intéressante avec au moins un insecte herbivore ont été extraites, fractionnées et testées par bioessai dans le but d'isoler l'élément chimique actif. Les insectes qui utilisent ces plantes comme plantes hôtes ont été collectionnés dans les forêts de la République de Panama. Par la suite, les insectes ont été analysés pour savoir comment ils métabolisaient les molécules toxiques de la plante. Dans cette étude, deux plantes ont été principalement étudiées avec leurs insectes correspondants: (1) Vismia baccifera et (2) Mikania guaco. L'étude d'une espèce générale et d'une espèce spécifique de lépidoptères a révélé qu'ils séquestrent, absorbent et concentrent la vismione B de leur plante hôte, la Vismia baccifera. Cette molécule est un élément cytotoxique actif contre trois lignées de cellules cancéreuses. L'étude de deux types spécifiques de coléoptères a révélé qu'ils séquestrent une nouvelle molécule, la guacanone, à partir de la vigne Mikania guaco. Cet élément a été isolé par l'auteur de cette étude. Il s'est révélé actif contre le Trypanosoma cruzi, l'agent responsable de la maladie de Chagas. Un type général de coléoptère ne s'est cependant pas révélé avoir les mêmes propriétés.
ii ACKNOWLEDGEMENTS
1 thank my supervisors Todd Capson and Tim Johns for guidance and encouragement.
1 thank Don Smith who served in my thesis advisory committee.
1 am grateful to Catherine Potvin for support in Panama.
1 thank Donald Windsor and Annette Aiello for their knowledge about insect ecology, collecting and rearing, and for the identification of several insects.
1 thank Ahmed Hussein for the e1ucidation of the structure of Guacanone and also for guidance in chemistry techniques.
1 am grateful to Maria HelIer and Erika Garibaldo for technical support at STRI and within the ICBG and Carolyn Bowes for administrative support from the department of Plant Science.
1 thank Rafael Aizprua, Nayda Flores, Blanca Arauz, for botanical assistance in the collection and identification of plant specimens.
1 thank Nivia Rios for assistance in the chemistry lab and Johant Lakey for instruction on HPLC techniques.
1 thank Eduardo Ortega, Luz Ramiro, Mahabir Gupta and their laboratories for running the disease bioassays.
1 thank Kerry McPhail at the University of Oregon for running Mass spectrometry for my samples.
Financial support for this project was provided by the National Institute of Health (NIH), the Organization of American States (OAS), and the Office Qœbec• Amériques pour la Jeunesse (OQAJ).
1 am extremely grateful to Dana, Enn, Karl, Rory as well as Scott Smith, Andrew Moeser, Neilan Kuntz and Javier Barrios.
iii CONTRIBUTIONS OF AUTHORS
The manuscripts were written by the first author under the guidance of the supervisor, Todd Capson (co-supervisor Tim Johns offered input but is not listed as an author on either paper). Ahmed Hussein contributed to the work described in the first manuscript as the chemist who isolated and elucidated the structures of the chemical compounds described therein. Ahmed Hussein contributed to the second manuscript by elucidating the structure of the compound isolated by the first author from the plant Mikania guaco. AlI other experiments were performed by the first author using materials, equipment and facilities supplied by the primary supervisor in
Panama.
iv TABLE OF CONTENTS
ABSTRACT ...... i
RÉSUMÉ ...... ii
ACKNOWLEDGEMENTS ...... iii
CONTRIBUTIONS OF AUTHORS ...... iv
LIST OF FIGURES ...... vii
1. INTRODUCTION ...... 1 1.l.Insects in bioprospecting ...... 1 L2.Relevance ofproject to science ...... 2 1.3. Broader relevance ofproject: health and conservation ...... 3 1.3.l.Irreplaceable sources ofnovel chemical structures ...... 3 1.3.2.Using drug discovery as a means ofpromoting forest conservation ...... 4 1.3.3.The impact of tropical parasitic diseases ...... 5 1.3A.American Trypanosomiasis (Chagas' Disease) ...... 5 lA.Details of study and methodologies used ...... 7 1.4.1.Study site ...... 7 lA.2.Criteria for choice of plant-insect associations ...... 8 1.4.3.Recollection of plants ...... 8 lAA.Collection of insects ...... 8 1.4.5.Rearing of insects ...... 9 2. REVIEW OF THE LITERATURE ...... 11 2.l.Insects in bioprospecting ...... 11 2.2.Plant secondary metabolites ...... 11 2.3.Chemical ecology ofplants ...... 12 2A.Plant-insect interactions ...... 13 2.5.Aposematism and other toxicity indicators ...... 14 2.6.Groups of insects that sequester plant compounds ...... 17 2.7. Compounds known to be sequestered by insects ...... 19 2.8.An insect's potential as a guide to novel medicines ...... 20 3. INTRODUCTION TO MANUSCRIPTS ...... 22 4. MANUSCRIPT 1 ...... 23 ABSTRACT ...... 24 4.1. Introduction ...... 25 4.2. Materials and methods ...... 31 4.3. Results ...... 33 4.4. Discussion ...... 37 5. LINKING STATEMENT ...... 40
v 6. MANUSCRlPT 2 ...... 41 ABSTRACT ...... 42 6.l. Introduction ...... 43 6.2. Materials and methods ...... 47 6.2.l. Plant and insects ...... 47 6.2.2. Bioassay-guided fractionation of Mikania guaco ...... 48 6.2.3. Insect-plant chemical ecology ...... 50 6.3. Results ...... 50 6.3.1. Compound isolation and elucidation ofstructure ...... 50 6.3.2. Insect-host plant chemical ecology ...... 51 6.4. Discussion ...... 53 7. GENERAL DISCUSSION & CONCLUSIONS ...... 55 8. LITERATURE CITED ...... 60 9. APPENDIX A: Bioassays ...... 67 9 .1.Cancer bioassay ...... 67 9.2.Trypanosoma cruzi bioassay ...... 67
vi LIST OF FIGURES
Figure 4.1. Structure of three anticancer compounds isolated from Vismia baccifera ...... 29 1. Vismione B 2. Desacetylvismione H 3. Desacetylvismione A
Figure 4.2. Photographs of host plant Vismia baccifera and two primary herbivores: Periphoba arcaei and Pyrrhopyge pseudophidias ...... 30
Figure 4.3. HPLC results comparing pure compound vismione B, crude extract of plant Vismia baccifera, and extracts of different life stages of Pyrrhopyge pseudophidias ...... 35
Figure 4.4. HPLC results comparing pure compound vismione B, crude extract of plant Vismia baccifera, and extracts of different life stages of Periphoba arcaei ...... 36
Figure 6.1. Photographs of host plant Mikania guaco and three primary herbivores: Echoma anaglyptoides, Platyphora ligata and Eugenysa coscaroni ...... 46
Figure 6.2. Chemical structure of Guaconone ...... 51
Figure 6.3. HPLC results comparing the purified compound Guacanone, crude extract of plant Mikania guaco, and extracts of three beetles that feed on the plant ...... 52
VB 1. INTRODUCTION
1.1. INSECTS IN BIOPROSPECTING
Tropical insects represent an enormous group of organisms that contain a great diversity ofunexplored organic compounds (Sittenfeld et al., 1999). Insects are especially well known for their diversity of defensive secondary compounds, most of which originate in the insect's host plant. These defensive compounds have similar structures to mole cules that are responsible for most pharmacological activity in plants (Sittenfeld et al., 1999). Almost all arthropods also have a complex gut and symbiont microbial biota that synthesize other organic molecules. Despite their wealth of potential compounds, there has been little incorporation of insects into the field of bioprospecting because of the difficulty in identifying insects in the field and the difficulty in collecting insects in large enough quantities for chemical extraction and use in disease bioassays (Sittenfeld et al., 1999). An International Cooperative
Biodiversity Group (ICBG) project in Costa Rica attempted to base their bioprospecting program on the isolation of medicinally-active compounds from arthropods. They failed to produce any interesting medicinally-active compounds mainly due to the above-mentioned problems.
An alternative method of utilizing insects in the search for pharmacologically active compounds is to use them as guides to plants of interest, from which compounds can then be easily isolated. The advantage is the ease with which scientists can gather large amounts of plant material to work with. Many insects selectively sequester secondary metabolites from their host plants in order to make use of their biological activity (Harborne, 1999). Different groups of insects have
1 been shown to sequester plant compounds for such uses as defence, mating and colouration (Harbome, 1999; Geuder et al., 1997). Chemical activity in one biological system often signaIs cœmical activity in other biological systems, and it has been suggested that aposematic insects advertising toxic compounds sequestered from their food plant might serve to guide the drug discovery process (Sittenfeld et al., 1999). Whether or not using insects as guides to pharmacologically-active plants is a feasible strategy is the contextual question of this master' s project.
1.2. RELEVANCE OF PROJECT TO SCIENCE
The link between insects and plant-derived medicines has not been well established, as very little research has focused on this relationship. This project will not demonstrate that insects can or cannot be used successfully as guides in the drug discovery process but will add to the body of information that can eventually be used to substantiate or reject this claim. The various studies incorporated into this project
also increase the diversity of chemical-ecological studies of insects and their host plants. Broader knowledge of the insect groups that sequester plant secondary
metabolites, as well as the types of compounds that are sequestered by insects will
further understanding of the evolution of însect and plant diversity.
In addition, this project involved the isolation of novel compounds active
against cancer celllines and Trypanosoma cruzi. This in itself is an important
contribution to science and human health.
2 1.3. BROADER RELEVANCE OF PROJECT: HEALTH & CONSERVATION
1.3.1. IRREPLACEABLE SOURCES OF NOVEL CHEMICAL STRUCTURES
Natural products have been and continue to be an important source of pharmaceuticals in use today. For example, it has been estimated that 37% of total pharmaceutical sales, and 45% oftoday's bestselling drugs, are from natural products and their derived molecules (Frormann & Jas, 2002; Grifo et al., 1997).
Approximately 60% of the agents in clinical trial for the treatment of cancer owe their origin to natural products (Cragg & Newman, 2000).
Natural products are of inherently greater chemical diversity and complexity than synthetic compounds because of the high frequency of asymmetric centers
(which gives molecules a 3-dimensional topology which is difficult to synthesize
(Waterman, 1998). They are also generally larger in molecular weight and have an
increased oxygen content, a greater number of ring structures, and a variety of
functional groups that are unrepresented among synthetic compounds. These factors
underlie the continuing importance of investigating natural products as sources of
novel medicines. Given the small percentage of tropical plants that have been
examined for medicinal properties, investigations of wild tropical plants should
pro duce many new medicines. From the perspective of drug discovery, the
disappearance of tropical forests would deprive humankind of an irreplaceable source
of chemical diversity (Terborgh, 1992).
3 1.3.2. USING DRUG DISCOVERY AS A MEANS OF PROMOTING FOREST CONSERVATION
Drug discovery has great potential as a means of promoting forest conservation, but only if there are explicit economic benefits for tre countries that have the forest resources (Capson et al., 1996). Traditionally, none of the revenues derived from the sales of drugs discovered from plants in tropical forests have returned to the countries with the forest resources. Thus, drug discovery has provided no fiscal incentives to promote forest conservation. For example, Madagascar receives none of the approximately 300 million dollars per year generated by vincristine and vinblastine, despite the fact that the drugs were originally discovered in a Malagasy plant. Costa Rica's ICBG pro gram did have agreements in place to allow economic benefits from the drug discovery process to flow into Costa Rica, however the pro gram was limited in its success (Sittenfeld et al., 1999). Such economic incent ives for biodiversity conservation are crucial in developing countries such as Costa Rica or Panama, which are usually burdened with external debt and face pressure from population growth. The Panama ICBG collects plants only from within national parks to make the link between conservation and drug discovery more explicit.
4 1.3.3. THE IMPACT OF TROPICAL PARASITIC D ISEASES
Along with its conservation goals, the ICBG drug discovery program also aims at increasing global human health. The ICBG fa;uses on testing plant extracts against tropical diseases as well as cancer and HIV. Tropical diseases continue to exact a huge toll in the developing world (Sachs, 2002). Collectively, malaria,
Chagas' disease and leishmaniasis affect 3 billion people, most of who survive on less than two dollars a day (Gelb & Hol, 2002). For many ofthese diseases, the
CUITent drugs available for treatment are inadequate, and the parasites are becoming drug resistant in many areas (Gelb & Hol, 2002). Most pharmaceutical companies have concentrated on finding medicines to treat the diseases of industrialized countries (e.g. cancer and heart disease) and there has been little development of new antiparasitic drugs since the 1960s (WHO, 2000). Beyond the mortality inflicted upon residents of developing countries, outbreaks of parasitic diseases are likely to affect rich countries as well, since drug resistant diseases can spread via immigration and travel (Kirchhoff, 1993).
1.3.4. American Trypanosomiasis (Chagas' Disea;e)
Trypanosoma cruzi is the causative agent of Chagas' disease, a chronic multisystemic disease that affects millions of people in temperate, subtropical, and tropical regions in the Americas and West Indies, killing 10-20% of the people it infects (Ge1b & Hol, 2002). Approximate1y 25% of Latin America's population lives
5 in areas at risk from this disease. Infection is transmitted to humans by bloodsucking insects of the family Reduvidae. The overall incidence of the disease has been estimated at 200,000 cases a year and over 70,000 individuals die annually as a consequence of the infection (WHO, 1991; Dusanie, 1991).
There are three clinical stages in Chagas' disease: a short acute stage, a long-lasting ehronic stage, and a long, asymptomatic stage (WHO, 1991). There is no evidenee that individuals in the ehronie or asymptomatie phases of the disease benefit from drug therapy, and therefore treatment is given only to patients in the short aeute phase of Chagas' disease. Treatment with nifurtimox (a nitrofuran derivative) and benznidazole (a nitroimidazole) results in adverse side effects, including weakness, anorexia, nausea, and vomiting in most patients (Viotti et al,
1994). Continued therapy is frequently associated with toxie hepatitis, and central and peripheral nervous system symptoms such as loss ofmemory, tremor polyneuritis and seizures. In one study, lymphomas developed in 33% of the rabbits treated with nifurtimox and 42% of the rabbits given benznidazole (Viotti et al,
1994). Thus, there is an urgent need to develop new medicines to treat Trypanosoma cruzi (Gelb & Hol, 2002).
6 1.4. DETAILS OF STUDY AND METHODOLOGIES USED
1.4.1. STUDY SITE
Most of the work for this project was carried out at the Smithsonian Tropical
Research Institute in Panama, within a group called the International Cooperative
Biodiversity Group (ICBG-Panama). The Panama ICBG emphasizes the participation of the Panamanian govemment and host-country scientific and conservation institutions. The ICBG has been collecting plants in Panama and testing them in various disease bioassays for four years and provided the ideal base of infonnation on tropical plants with novel phannacological activity with which to begin this study. Panama is a well-known hotspot ofbiological diversity, connecting the South American continent to the rest of the Americas.
The plants included in this study were collected in Panamanian National Parks and tested at Florida State University (Panama campus) for activity against three cancer celllines (breast cancer, lung cancer and cancer of the Central Nervous
System), HIV, malaria, leishmaniasis and trypanosomes (the causative agent of
Chagas' disease) (disease bioassays are described in Appendix A). In two different chemistry laboratories at tre University of Panama, extracts from plants that showed activity against one or more of the diseases mentioned above were purified in order to find the active component. This process was also carried out at STRI using High
Perfonnance Liquid Chromatography.
7 1.4.2. CRITERIA FOR CHOICE OF PLANT-INSECT ASSOCIATIONS
• the plant species had to be consistently active in one of the disease bioassays • the plant species had to be relatively common • the insect species had to be clearly associated with the plant species
1.4.3. RECOLLECTION OF PLANTS
After choosing plant candidates that showed initial activity against a disease and that had an association with an aposematic in sect, these plants were recollected from within the national park it was originally collected from. Any collections made from other areas were tested in the bioassays to confirm activity of the local population. Approximately 20g of extract were needed to isolate active components from a plant but this was variable depending on the species. The amount of leaves needed to acquire this amount of extract was also variable but could be calculated after the first collection and extraction of a plant.
1.4.4. COLLECTION OF INSECTS
There was little ecological information available for the species of insects that were included in this study. What information existed was gathered from local entomologists and from two large insect collections - at the University of Panama and at STRI. Insects were extremely heterogeneously distributed in time and space
8 and constant monitoring of insect host plants was necessary to collect a sufficient quantity of material for chemical analysis. Although the host plant of each insect species being collected was known, little else was known about these insect species, including peak times when they would appear in large numbers. Peak collection times for many insects are July (emergence of the first generation during the rainy season) and October (peak of the second rainy season generation) and therefore partic ular collecting effort was made during these times. Insects were located by haphazard and directed search, and a monitoring schedule was established for every plant species.
Between 20 and 40 individuals of each host plant species was identified.
After this population was established, it was visited on at least a weekly basis to monitor for insect activity. Any insect found feeding on the plant species was collected and brought back to the lab for rearing.
1.4.5. REARING OF INSECTS
Insects were reared according to protocol established by STRI staff entomologist
Annette Aiello. Twenty cages were constructed using wire screening, Petri dishes and masking tape. One adult of each insect species was mounted for identification and reference purposes.
The goal of rearing was to secure a sufficient quantity of each life stage of the insect being collected. When the insect being reared reached the life stage that was needed for chemical extraction, the individual was starved for 36 hours to rid the guts
9 of contents that could be confounding in chemical analysis, and then frozen. AlI shed larval skins, feces and insect specimens were kept in methanol at -80°C until needed.
Voucher specimens for all insects were deposited in two insect collections at STRI -
Coleoptera in Don Windsor's collection, Lepidoptera in Annette Aiello's collection.
10 2. REVIEW OF THE LITERATURE
2.1. INSECTS IN BIOPROSPECTING
Tropical insects represent a little-explored source of a great diversity of organic compounds (Sittenfeld et al., 1999). Most of the compounds found in insects are sequestered from their host plants, which explains the diversity and complexity of these secondary metabolites. The pharmaceutical industry is interested in similar compounds for use as human medicines, pesticides and other products. This has led to programs such as the ICBG in Costa Rica collecting and testing insects in disease bioassays. The work in this thesis investigates an alternative method of utilizing insects in the search for pharmacologically-active compounds, which is to use them as guides to plants of interest from which compounds can then be isolated with greater ease. This strategy is based on theory regarding plant and insect chemical ecology.
2.2. PLANT SECONDARY METABOLITES
An aspect of metabolism which distinguishes the plant and animal kingdoms is that plants contain pathways that branch off from primary metabolism, which are not essential to the survival of the organism (fungi haw this characteristic as weIl).
The products of the se peripheral pathways are called 'secondary metabolites' and have been defined as 'end-points ofmetabolism with no strictly defined function'
(Edwards & Gatehouse, 1999). Thousands of secondary products have been described from higher and lower plants. The motivation to characterize plant
11 secondary metabolites is often driven by commercial interests, as they have been the source of many valuable drugs, pesticides and chemicals important to the food industry (Caporale, 1995; Edwards & Gatehouse, 1999).
2.3. CHEMICAL ECOLOGY OF PLANTS
There are many projects throughout the world that are dedicated to finding economicaUy relevant chemical compounds, most concentrated in the biodiverse tropics. Finding these compounds within the enormous diversity of life on the planet is equivalent to finding a needle in a haystack. The field of ethnobotany has utilized the knowledge of indigenous peoples to identify plants of medicinal interest, however more recently this field has been fraught with controversy revolving around inteUectual property rights. The botanists in the Panama ICBG bioprospecting project use ecological the ory to direct their searches for active plant compounds. For example, they began by collecting young leaves separately from the mature leaves of
aU plant species, foUowing the theory that soft and expanding young leaves in tropical
forests are more susceptible to herbivory and therefore must utilize more secondary metabolites to protect themselves from herbivores. In contrast, older leaves are
defended by medicinaUy uninteresting tannins and toughness (Coley et al., 1985;
Coley & Aide, 1991). Young 1eaves have been shown to have higher concentrations
of alkaloids than mature leaves. In fact, it has since been shown that significantly
more young leaves have been active in disease bioassays within the Panama ICBG
12 than mature leaves (Kursar et al., 1999). With these preliminary findings, the ICBG has been making use of the theory that many secondary metabolites will occur in higher concentrations in young leaves and novel compounds that are active in disease bioassays may be more abundant in young 1eaves.
The 'young leaf' theory has yielded interesting results in the Panama ICBG.
However, botanists must still go into the forest and collect samples randomly from aU the plants that they encounter there. Insect ecology could theoreticaUy narrow down the possibilities of plant species that could be collected and tested in disease bioassays. Programs like the ICBG are often faced with many crude extracts that are active to sorne degree and must decide which plants to pursue for compound isolation using bioassay-guided fractionation. An alternative use ofinsects would be to investigate which pharmacologicaUy-active plants have associations with interesting insects and to use this information as criteria for choosing which preliminarily-active plants to pursue with bioassay-guided fractionation.
2.4. PLANT-INSECT INTERACTIONS
The observation that many species of phytophagous insects feed on only one, or a few, host plant species, led Ehrlich and Raven (1964) to propose a theory of co evolution in which secondary metabolites in plants play an important role in determining the specificity of interaction between insects and their host plants. The specific predator(s) of a given plant species were suggested to have adapted to astate
13 where they were unaffected by secondary compounds present in the plant as a defence against non-adapted insect predators. The diversity in plant species could then be partly explained by the need for plants to develop different types of biochemical defence against insect predators, resulting in a high level of diversity in secondary compounds. In response, insect species would themselves diversify, with individual insect species developing specific biochemical processes to overcome the effects of the defence compounds of their chosen host. This progression has been referred to as a 'biochemical arms race' by sorne authors. The co-evolution theory would explain the incredible diversity of plant species (more than 250000 species), secondary compounds (more than 30 000 structures characterized) and insect species
(an estimated 2-5 million) (Edwards & Gatehouse, 1999). It must be noted that 'co evolution' does not explain aIl plant-insect interactions. Plants do not produce secondary compounds solely as a defence against insect predators, and many insects which feed on plants are polyphagous.
2.5. APOSEMATISM & OTHER TOXICITY INDICATORS
The interest in attractive aposematic insects has led to many studies on insect sequestration of the toxins that these insects are advertising. Aposematism was first defined by Poulton (1890) as "an appearance which wams off enemies bccause it denotes something unpleasant or dangerous, or which directs the attention of an enemy to sorne speciaIly defended or merely non-vital part, or which wams off other
14 individuals of the same species." CUITent definition links an unpleasant or toxic quality of a prey (unpalatibility) with an advertisement ofthis feature (warning colouration) (Bowers, 1993). U npalatable insects are thought to advertise their defence by conspicuous colouration, gregariousness and sedentary behaviour
(Bowers, 1993). These characteristics of aposematic insects are the visual cues that highlight the plant-insect 'arms race' and constitute what could be used as a guide to pharmacologically-interesting compounds.
In sorne cases the link between aposematism and toxicity is very clear. It was found in several species from two moth families that when the larvae and adult are aposematic, both have toxic iridoids, but when the adult is not aposematic, most of the iridoids are emitted with the meconium upon eclosion (Boros et al., 1991).
However, many unpalatable caterpillars are not aposematic and sorne brightly coloured insects are not toxic (Bowers, 1993). In fact there are whole mimicry complexes containing many organisms where only a few individual species or even just one actually contain toxic defence compounds. Therefore aposematism is not a certain guide to biologicaUy active compounds and there are a lot of gaps in our knowledge of tropical insect-plant relationships that must be filled before we can come to any conclusions about the usefulness of using insects in the field of bioprospecting.
As many criteria as possible should be used to determine whether an insect is
advertising plant-derived defences. The 'arms race theory' suggests that insects that
specialize on only one plant species (or a smaU number of c10sely related plant
species with similar metabolic products) would be a more effective guide to toxic
15 compounds than generalist species, as they would have had the opportunity to evolve resistance to the most complicated and specifie of plant secondary metabolites.
However insects that initially developed the ability to sequester defensive compounds through monophagy have been found in sorne cases to be able to sequester toxins from other sources, and therefore generalist species cannot be discounted completely
(Duffey & Scudder, 1972). Insects that are apparent, aggregate, sedentary and long lived are those which would be most likely to bene fit from the advertisement of real defensive compounds and should be the most useful as indicators of active plant compounds. However these criteria need to be investigated in more depth in the context of bioprospecting.
It is also important to study the differences in chemistry between the life stages of each insect species. It has been found that different life stages of an insect species can contain different types and quantities of defensive compounds (Pasteels et al., 1983). Adults in the Chrysomelid beetle Oreina gloriosa were found to have a more complex mix of cardenolides than immature stages with a broader polarity spectrum (Eggenberger & Rowell-Rahier, 1995). In sorne beetle species where larvae and adults eat the same plant, they employ different compounds for defence. This could be a result of different levels of aposematism and thus different predators feeding on the different life stages. There are sometimes even differences in chemical defence between the various larval instars of a species (Pasteels et al., 1983).
The immature forms of sorne Chrysomelid beetles protect themselves with
"shields" of fecal residues that are thought to contain toxic chemicals that they acquire from the plants they eat. It is assumed, but unproven, that these shields have
16 potent plant chemicals that defend them from predators (Venc1 et al., 1999). Beetle
1arvae that uti1ize feca1 shields are thus another potentia1 indicator of active plant compounds.
Other ecologica1 findings cou1d be used strategically in later stages of the drug discovery process. For example, Însects can se1ective1y sequester sorne compounds and not others. A Lycaenid butterfly was able to sequester preferred kaempfero1 flavonoids and not other flavonoids, possib1y by selective glucosy1ation (Wiesen,
1994). A hymenopteran species was able to na vigate the toxicity of various veratrum a1kaloids in its host plant in four ways. One alkaloid was metabolized and acetylated, another was taken up as is, another was degraded and another was excreted intact
(Schaffner, 1994). This know1edge cou1d be used to determine which compounds are most usefu1 to humans as drugs, possibly indicating different leve1s of cytotoxicity or different mechanisms of activity. Any new compounds, slightly changed from their original states by the insects themse1ves, could offer new leads to the pharmaceutical industry searching for nove1 chemica1 structures.
2.6. GROUPS OF INSECTS THAT S EQUESTER PLANT COMPOUNDS
Sorne groups of insects are more likely to sequester toxic compounds than others. Previous studies on insect sequestration have focused on the orders
Lepidoptera and Coleoptera, but examp1es of insects sequestering plant toxins have
17 also been found in Orthoptera, Hemiptera and Homoptera (Williams et al., 2001;
Duffey & Scudder, 1972). However chemical defence is very unevenly distributed even within genera (Pasteels et al., 1983). As mentioned, trends that exist seem to function more on an ecologicallevel than a phylogenic level. For example, larger, long-lived insects, and insects that live in exposed areas (i.e. pasture, gaps, where both the plant community and other ecological conditions are distinct from forest interiors) are more likely to develop chemical defence (Pasteels et al., 1983).
It still holds, however, that an insect belonging to a taxon well-known to inc1ude plant compound-sequestering insects is more likely to be use fuI as an indicator than an insect from a group with no record of sequestration. Chrysomelid beetles have recently become a focus of studies on insect defence. Exocrine chemical defence is common in the Chrysomelid leafbeetles (Pasteels et al., 1992). They have been shown to both sequester N-oxides in specialized exocrine glands as well as produce mixtures of cardenolides that are not found in their food plants but synthesized œnova from phytosterols. Defensive chemicals have been found both in the exocrine glands and in the bodies ofmany beetles from this family. Other examples of insect families known to sequester plant compounds are Saturniidae,
Ithominae and Arctiidae (m oths), and Curculinoid beetles (Harborne, 1999; Hartmann etaI., 1999).
18 2.7. COMPOUNDS KNOWN TO BE S EQUESTERED BY INSECTS
Previous studies examining insect use of plant secondary metabolites have concentrated on certain classes of compounds. Studies of the Monarch butterfly
(Danainae) led to the discovery that these butterflies were sequestering cardiac glycosides from their asclepiad host plants as well as sequestering pyrrolizidine alkaloids as adults (Boppre, 1986). This is still the classic mode 1 of insectlplant biochemical coevolution, but it is now known that species from at least 32 genera of
Lepidoptera sequester pyrrolizidine alkaloids CV on Nickisch-Rosenegk & Wink,
1993; Wink & Schneider, 1988). This class ofalkaloids has been more studied than any other defence group mostly because of its importance to humans as a frequent poison consumed by vertebrates, including cattle.
Aside from the Danainae family, other families of Lepidoptera are well known for their ability to sequester toxins. Still only a small fraction of arthropods have been examined for their ability to secrete defensive compounds. These studies have been concentrated on specific groups of insects and have neglected most of the enormous diversity of insects. Because of this, only a small array of secondary metabolites has been found to be sequestered by insects. Aside from pyrrolizidine alkaloids, common defence compounds found have included cardiac glycosides, flavonoids, quinolizidine alkaloids, veratrum alkaloids, piperidine alkaloids and carboline alkaloids. Defensive compounds include non-specifie irritants acting as repellents and true poisons that act at specific sites or interfere with specific physiological processes. This second group includes heavier compounds than the
first class and includes the cardenolides, bufadienolides, cantharidin, quinazolinone
19 alkaloids and most of the other alkaloids (Pasteels et al., 1983). A more comprehensive survey of insects and their defensive compounds has to be carried out to get a more accurate picture of the diversity of compounds being sequestered by insects.
2.8. AN INSECT'S POTENTIAL AS A GUIDE TO NOVEL MEDICINES
An additional feature of importance for a natural products-based screening program, especially one guided by ecological insight, is the evolutionary history of the compounds. Because the molecular structures of natural products have been moulded over thousands of years in response to intense interactions among species, they are ideal for maximizing the success of screening for novel, complex structures, and for identifying previously unrecognized target proteins or activities (Harbome,
1988). An ecologically-guided collection and bioassay pro gram using insects will take advantage of the unique and specific histories of co-evolved plants and insects and could lead to many interesting discoveries in the field of natural products.
For insects to work as guides to pharmacologically active compounds, they need to be sequestering the same types of compounds that are successfully used as medicines. There is a host of criteria that has to be met in order for insects to be used successfully as guides to potential medicines. Many defence compounds in beetles are volatile, and would therefore be of no use as medicines. Compounds that are too cytotoxic might cause more damage than benefit to humans. Similarly, broad-
20 spectrum toxins might cause problems ta human health and therefore selectivity is a criterion that is desired in compounds destined for use in the pharmaceutical industry.
The literature has demonstrated that there is an overlap between classes of compounds used in defence by insects and classes of compounds use fuI to the pharmaceutical industry. Quinolizidine alkaloids come from a wide variety of plants, although are concentrated in the family Fabaceae. A medicinal example is sparteine which has been used as a cardiac stimulant and oxytocic agent (Sim, 1965). This class of compound has been shown to have been sequestered Genista acanthoc/ada, and associated parasite and several other insects (Wink & Witte, 1993; Szentesi &
Wink, 1991; Montllor et al., 1990; Stermitz et al., 1989). Another class ofalkaloid that has been sequestered by insects is veratrum alkaloids which are steroidal alkaloids that possess hypotensive action through reflex inhibition of pressor receptors in the heart and carotid sinus (Sim, 1965; Schaffner, 1994). A third interesting example is the case of the Urania moth and its host plant, the genus
Omphalea (Euphorbiaceae). Sugar-mimicking alkaloids isolated from the plant genus were found to be active against HIV by researchers at Kew Gardens in England
(Schulz, 1990). Urania moths are stunningly colourful day-flying maths that migrate in conspicuous groups and feed specifically on the host plant. The same plant compounds that are active against HIV were found to have been sequestered by the moth from its host plant (Kite et al., 1997).
21 3. INTRODUCTION TO MANUSCRIPTS
Two manuscripts make up the main body of this thesis. During the course of the study, several plant-insect associations were investigated. The investigations of the plant-insect associations centered on the plants Vismia baccifera and Mikania guaco represent the most complete studies. Interesting and comp1emet1ary resu1ts were found from studies of:
1. Mabea occidentalis and associated butlerflies of the genus Unica.
2. Hasseltiafloribunda and its only associated phytophagous predator
Plagiodera viridimaculata.
These studies could be continued in the future and would add more substance to the body of work inc1uded here within. The first paper (Manuscript 1) was a combined effort by the primary author and a post-doctorate chemist from the
University of Panama to investigate the relationship between a plant, its activity against cancer, and the aposematic insects that feed on this plant. The first study served as a leaming too1 for the first author to be able to complete all sections of the second study (Manuscript 2), inc1uding isolation of the active components of Mikania guaco.
22 4. MANUSCRIPT 1
Sequestration of cytotoxic vismiones by Pyrrhopyge pseudophidias (Lepidoptera: Hesperiidae), Periphoba arcaei (Lepidoptera: Saturniidae) and a Chrysomelid beet!e (Coleoptera: Chrysomelidae) from the host plant, Vismia baccifera.
ClARA RAUDSEPP-HEARNEY AHMED HUSSEIN3 and TODD L. CAPSONJ,2*
1Smithsonian Tropical Research Institute Apartado 2072, Balboa, Ancon, Republic of Panama
2Department of Plant Science Mc Gill University 21,111 Lakeshore Road Ste. Anne de Bellevue, Quebec H9X 3V9, Department of Plant Science, McGill University
3Centro de Investigaciones Farmacognosticas de la Flora Panamefia (CIFLORPAN), Facultad de Farmacia, Universidad de Panama, Apartado 10767, Estafeta Universitaria, Panama, Republica de Panama, University of Panama
*To whom correspondence should be addressed. E-mail: [email protected]
23 ABSTRACT
The insects Pyrrhopyge pseudophidias (Lepidoptera: Hesperiidae), Periphoba arcaei (Lepidoptera: Satumiidae) and a Chrysomelid beetle (Coleoptera:
Chrysomelidae: subfamily Eumolpinae) were investigated for their ability to assimilate the compounds vismione B (1), deacetylvismione H (2) and deacetylvismione A (3), cytotoxic compounds previously isolated from tre host plant,
Vismia baccifera. The two lepidopterans were examined from Jd instar to adult while the beetle was analyzed only at the adult stage. Compound 1 was found in both the third and fourth in stars of Pyrrhopyge pseudophidias but not in the Sh instar, the pupa nor the adult. Compound 1 was found in the :td and 3rd instars of Periphoba arcaei but not in the pupae or adult. Compound 1 was also present in the shed larval skins of
P. arcaei suggesting that the compound might be sequestered for defensive reasons.
Compounds 1 and 3 were found in the adult stage of the Chrysomelid beetle.
Key Words - Pyrrhopyge pseudophidias, Periphoba arcaei, Vismia baccifera, Lepidoptera, Coleoptera, vismiones, insect-plant interactions, sequestration.
24 4.1. INTRODUCTION
Many insects selectively sequester secondary metabolites from their host plants for uses such as defence, mating and colouration (Harborne, 1999; Geuder et al., 1997). It has been suggested that aposematic insects that assimilate toxic compounds from their food plants might serve to guide the drug discovery process
(Sittenfeld et al., 1999). In practice, however, the authors of a recent study reported
"the most important practical problem has been the relatively small amount of crude extract available to pursue bioassay-guided fractionation" (Sittenfeld et al., 1999). To avoid problems inherent in the isolation of chemical compounds from insects, we have pursued a strategy in which aposematic insects are used as guides to plants with compounds with potential therapeutic applications. The research is carried out as part of the activities of the Panama International Cooperative Biodiversity Groups
Pro gram (ICBG), a project directed towards the discovery of treatments of cancer and tropical diseases from tropical marine and terrestrial ecosystems (Mendoza et al.,
2003; Hussein et al., 2003). We also sought to answer the complementary question as to whether compounds with cytotoxicity against tumour celllines were also assimilated by the aposematic insects in question. Only if insects are found to preferentially sequester the same compounds that are active against certain diseases could they be used as indicators of potential new medicines.
Plants from the genus Vismia have been shown to produce a range of
secondary metabolites, inc1uding triterpenoids, prenylated anthrones, anthraquinones,
bianthraquinones, benzophenones, and lignanes (Delle Monache, 1983; Arauji et al.,
1990). Among the compounds assimilated by Vismia spp. are the vismiones,
25 anthtanoids which have been isolated from plants in the tribe Vismiaea (Clusiaceae), inc1uding the genera Vismia, Harungana and Psorogpermum (Cassinelli and Geroni,
1986). The vismiones have been shown to have a range of activities inc1uding cytotoxicity (Cassinelli and Geroni, 1986; Seo et al., 2000) and antifeedant activities
(Simmonds et al., 1985). V. baccifera is a common shrub in Panama, abundant in older clearings and forest edges between sea level and 1000 meters (D'Arcy, 1987).
The shrub is of variable height (2-22m) with characteristic tomatose, orange branches and abundant sap that is secreted from all vegetative structures (Robson, 2001).
Flowers are produced twice a year and young leaves are flushed continually throughout the year (D'Arcy, 1987). We recently reported the isolation ofthree compounds 1, 2 and 3 from Vismia baccifera following cytotoxicity against the NCI
H460 (lung), SF268 (CNS), and MCF7 (breast) celllines (Figure 4.1.; Hussein et al.,
2003). Certain vismiones have been shown to haw good antifeedant activity against
Lepidoptera larvae and Orthoptera (Simmonds et al., 1985). Both compounds a 1 and
3 showed deterrent activity against Lepidoptera larvae, but 1 was consistently more active than 3, showing 2.4-fold more potency against Spodoptera exempta
(Simmonds et al., 1985). The assimilation of any of the compounds had not been previously demonstrated.
We studied the ability of two different Lepidoptera and one Coleoptera to assimilate highly cytotoxic vismiones known to be active against three different cancer cell lines, NCI-H460 (lung), SF268 (CNS), and MCF7 (breast) (Monks et al.,
1991). Both Lepidopterans are known to utilize Vismia baccifera (Triana & Planch)
as a ho st plant. We had previously shown that V. baccifera is a source of three
26 cytotoxic compounds, vismione B (1), deacetylvismione H (2) and deacetylvismione
A (3) (Hussein et al., 2003). The most active of the three compounds, deacetylvismione H, showed ICso values (the concentration of extract required to inhibit 50% of the celllines) of 400, 600 and 600 ng/mL in the breast, lung and CNS celllines, respectively.
Regular monitoring of populations of V. baccifera in different localities yielded a total of three insects feeding on this species in quantities sufficient for extraction. The insects were identified by comparing individuals with voucher specimens previously collected by STRI entomologists. The three insects included in this study were Pyrrhopyge pseudophidias Bell, 1931 (Lepidoptera: Hesperiidae),
Periphoba arcaei Druce, 1886(Saturniidae: Hemileucinae) and one beetle
(Chrysomelidae). The Chrysomelid beetle is part of the subfamily Eumolpinae, is bright orange in colour and is highly exposed while feeding and which, to date, it has only been observed on Vismia spp. Each of the three insect species is aposematic and extremely visible on the leaf surface to potential predators. The insects' bright appearance may signal the presence of defensive chemical compounds within the insect that are either biosynthesize d or sequestered from the host plant (Bowers,
1993).
Pyrrhopyge pseudophidias is a skipper butterfly (Family: Hesperiidae) that specializes on young leaves of the genus Vismia and occurs from Panama to Brazil
(Evans, 1951). In Panama the larvae have been found on both V baccifera and V billbergiana. Pyrrhopygines of this colouring are part of a vast group of aposematic, mimetic caterpillars (Burns & Janzen, 2001). Pyrrhopyge pseudophidias in the larval
27 stage is bright red with yellow striping and fine white hairs projecting outwards from the body. The adult butterfly is less aposematic, black, white-fringed, with a white stripe tapering across the forewing from below the mid-costa to above the tomus, and red-orange on both its head and rump. The larvae feed exposed on the leaf, but when not feeding and while in pupal form are enc10sed in a leaf shelter.
Periphoba arcaei (Druce, 1886) is in the family Satumiidae (subfamily:
Hemileucinae) and ranges from Mexico to Colombia (Janzen, 1982). P. arcaei are generalists and have been found and reared in Panama on both young and old leaves of V baccifera, Quassia amara, Anacardium excelsum, and in Costa Rica on at least
8 different plant families (Daniel Janzen, unpublished rearing records). The species has gregarious larvae, sedentary behaviour and conspicuous colouration, a suite of characteristics that strongly suggests unpalatibility and the presence of biologically active secondary metabolites (Bowers, 1993). First and second instar larvae are bright red. Subsequent instars are green with red lateral stripes, and the s:h instar develops a bright pink colouration prior to pupation. AlI instars have many branched spines. The adult moth is variable in appearance but non-aposematic with a dull brown colouring.
Larvae remain aggregate and sedentary upon the leaf surface for up to 60 days.
28 1.
OH 0
10 OH Vismione B
2. OH OH
15 1
\~O 10 OH /-- ~I
14 Deacetylvismione H
3.
\0 OH
Deacetylvismione A
Figure 4.1. Three cytotoxic vismiones isolated from the species Vismia baccifera
29 b
a c Figure 4.2. (a) Vismia baccifera, (b) Periphoba arcaei, (c) Pyrrhopyge pseudophidias
30 4.2. MATERIALS AND METHODS
Inseet material. Insects were collected from individuals of their host plant
Vismia baeeifera within three national parks in the Republic of Panama (see Figure
4.2.). Collections occurred between April and August of 2002, during which time individual trees were visited regularly to check for any insects feeding on the species.
Pyrrhopyge pseudophidias was collected from V baeeifera at Barro Colorado Natural
Monument, Chagres National Park and Altos de Campana National Park. Periphoba areaei was collected as an aggregation of 8 larvae from V baeeifera in Altos de
Campana National Park on June 27, 2002. Chrysomelid beetle individuals were collected from Chagres National Park and Altos de Campana National Park.
Rearing ofinseets. AIl insects were raised in wire cages on moist paper towel and cut foliage of V baeeifera was provided in excess. Cages were kept in Ziploc bags to maintain humidity levels. Each day, larval frass was removed and the paper towel was changed. For chemical analysis, instars, pupae, and adults were killed by deep-freezing (-80°C) and then stored in methanol at the same temperature. Larvae
were kept without food for at least 36 hours prior to deep-freezing to ensure that the
gut was empty. Additionally, sorne samples were dissected to remove the gut
completely to control for potential food plant residue from gut contents. Adults were
frozen after they had discharged their meconiums. Samples of larval frass and shed
larval skins were collected and stored.
31 Food plant. Leaves of V. baccifera were collected in the vicinity of Panama
City, and from the national parks from whichthe insects were collected. Voucher specimens were deposited in the herbarium at the University of Panama (voucher number PMA 51365). The identity of V. baccifera was confirmed by Professor
Mireya Correa of the Smithsonian Tropical Research Institute and the University of
Panama.
Extraction. Insects and plants were homogenized with 5mL of cold methanol for 5 minutes with a mortar and pestle followed by treatment with a Polytron homogenizer at low temperature (Brinkman Instruments) for two minutes or until the mixture was homogeneous. The mixture was filtered under vacuum through
Whatman #4 filter paper and the marc was then washed with 50 mL of methanol. The mixture was then filtered through Whatman #1 filter paper. The extract was concentrated by rotary evaporation and stored at -80 oC.
Chemical analysis. Purified compounds 1-3 were isolated from the V. baccifera were obtained as described previously (Hussein et al., 2003). Insect-derived compounds that were shown to have the same retenti on times and UV spectra as plant-derived samples were then tested by mass spectrometry on a Kratos MS50TC
instruments to confirm their structure. HPLC analyses were carried out on a Waters
instrument (Milford, Massachusetts) with a 600 E Quatemary pump with a
photodiode array detector. HPLC analyses employed Cl8 Nova Pak columns
32 [analytical: 4.6x 250mm, 4 micron; preparative: radial compression module kit,
Nova-Pak, two 25 mm segments] with linear gradients ofCH3CN and H20.
4.3. RESULTS
HPLC analysis of cytotoxic compoundsfrom V. baccifera. Compounds 1-3 previously isolated from V. baccifera (1, 2, and 3) (Hussein et al. 2003) were purified by HPLC from a methanol/ethyl acetate extract of the plant. The relative quantities were 15: 5: 1 for compounds 1, 2 and 3, respectively. The ratio of compounds 1-3 reflects their relative abundances in MeOH extracts of plants or insects based on the peak areas by HPLC analysis when measured at 254 nm.
Analysis ofPyrrhopyge pseudophidias (see Figure 4.3.). The life stages of
h Pyrrhopyge pseudophidias that had sufficient material for analysis were Jd instar, 4 instar, gh instar, pupa and adult, in addition to frass. Compounds detected in the any of the insect's life stages or in the frass were compared with authentic standards, initially by comparison with retenti on time by HPLC and UV data followed by confirmation by mass spectrometry. Each of the compounds 1, 2 and 3 were present in the frass. Third and 4h instar larvae of P. pseudophidias contained 1. To ensure that the presence of 1 was not an artefact of residual food plant in the insect gut, insects were starved for 36 hours prior to analysis and in sorne cases, the digestive tract was completely removed. In both cases, compound 1 was present in comparable quantities. HPLC analysis of frass from the larvae of P. pseudophidias revealed a
33 similar pattern of compounds compared to the leaves of V baccifera. There were no detectable quantities compounds 1-3 in the Sh instars, pupae or adults.
Analysis ofPeriphoba arcaei (see Figure 4.4.). The life stages for which
h sufficient material for analysis were Jd instar, 4 instar, pupa and adult. As shown for
Periphoba pseudophidias, the feces of the larvae contained compounds 1-3, and in relative proportions similar to V baccifera. Compound 1 was found in the larval stages, but not in the pupae or adult. The other compounds were not present in any of the life stages. Compound 1 was also found in the shed larval skins.
Analysis ofthe Chrysomelid beetle. The adult stage contained both compound 1 and compound 3 in small quantities. Results were not satisfactorily conclusive.
34 a. Purified Compound Vismione S
,,,1. 1 j to.oo.. 1500 2000 2600 3000 MonLM5
b. Crude extract Vismia baccifera 01 ~u:.10.00 20.00 30.00 40.00 c. 3rd instar Larvae Pyrrhopyge pseudophidias
5.00 10.00 15.00 20 00 25.00 30.00 35.00 40.00 '~L"".,~~JMinutes adult d. Pyrrhopyge pseudophidias
Figure 4.3. HPLC traces of (a) vismione S, b) crude extract of Vismia baccifera, (c) 3rd instar larve of Pyrrhopyge pseudophidias,(d) adult of Pyrrhopyge pseudophidias. Observed at 254 nm.
35 a. Purified Compound Vismione B b. Crude extra ct Vismia baccifera
~LLL10.00 .20 00 30'00 40.001 c. Feces Periphoba arcaei
d. 0.1~ 0.1~ 01~ 3rd instarLarvae ~ oo~ Periphoba arcaei 0.0" ::~4:;:;:;::;::;::;:~~~~~Ac~::;;:;;:;;;;:;:;;;:;:;::;:;~ 15.00 20.00 25.00 30.00 35 00 40.00 Minutes
Figure 4.4. HPLC traces of (a) vismione B, (b) crude extract of Vismia baccifera, (c) frass of Periphoba arcaei, (d) 3rd instar larvae of Periphoba arcaei. Observed at 254 nm.
36 4.4. DISCUSSION
Of the three insects found on Vismia baccifera each was found to assimilate compound 1 from its host plant, Vismia baccifera. Pyrrhopyge pseudophidias and
Periphoba arcaei contained no detectable quantities of compounds 2 or 3 while the
Chrysomelid beetle also assimilated compound 3. Compound 2 was not detected in any of the insects. The heavy rcedation upon caterpillars in tropical forests (Dyer &
Coley, 2002) combined with the cytotoxic nature of compounds 1-3 suggests that the compound are likely to be sequestered by Pyrrhopyge pseudophidias and Periphoba arcaei for defensive purposes, an observation consistent with the observed anti feedant properties of 1 (Simmonds et al., 1985). The observation that compound 1 was assimilated by all three insects and showed 2.4-fold more anti-feedant activity against certain Lepidoptera than 3 is particularly interesting, and suggests the ability to of the insects to tolerate 1 may be particularly relevant to their adaptation to the host plant. While each of the compounds 1-3 showed significant cytotoxicity against a panel ofthree tumor celllines (Hussein et al., 2003), compound 2 showed an average of 4.3-fold greater cytotoxicity than 1 in three different tumor celllines. Thus there is no c1ear correlation between the tumor cellline-based cytoxicity and the assimilation of the compounds by the insect. Compound 2 is present in 3-fold higher concentrations in the host plant V baccifera than compound 1 suggests that the
assimilation of the latter compound is due to its chemical and/or biological properties
rather than the relative abundance in the plant. Chemically, the most obvious
chemical difference between 1, 2 and 3 is the fact that the isoprenyl side chain in 1 is
37 attached to C-8 via an oxygen bridge whereas the isoprenoid side chains in 2 and 3 is attached only at C-6 and C-7, respectively.
In Periphoba arcaei, compound 1 was also detected in the shed larval skins.
P. arcaei larvae survive in an aposematic aggregation for up to 60 days. In addition to· the apparent chemical defences, P. arcaei larvae also have spines which are also presumably used to protect the larvae from predation. While the larvae of Pyrrhopyge pseudophidias remains inside a leaf house when not feeding, the skipper butterfly larva is highly aposematic and is likely to be chemically defended. P. pseudophidias is a specialist on the genus Vismia. Specialists with narrow host ranges are often much more resistant to the effects of defensive compounds in their host plants than are generalists (Rosenthal & Janzen, 1979). Nevetheless, we observed in this study that both generalist (Periphoba arcaei) and the specialist (Pyrrhopyge pseudophidias)
Lepidopterans as well as the Chrysomelid beetle sequester the same plant compound
1, vismione B, suggesting that the chemical and/or biologie al properties are particularly appropriate for chemical defence (e.g., enhanced assimilation by the insect or toxicity).
In both Lepidopterans, 1 was present in the larval instars, but not in the adult.
Other studies have also shown that toxic substances that are found in aposematic stages are not present in otrer stages which do not have waming colouration (Boros et al. 1991), which appears to be the case in this study. It has been proposed that changes in levels of toxic compounds at different life stages may be due to different
ecological and evolutionary fcrces acting on the different life stages (Bowers, 1993).
Pupae are the most immobile and incapable ofbehavioral defence while larvae are
38 much more sedentary than the winged adults. Pyrrhopygine butterflies have been recorded as having a very low rate of ptrasitization, which is also consistent with chemical defence (Burns & Janzen, 2001). The potential autotoxic effects during metamorphosis, when massive reorganization of the internaI organs, and high levels of cell growth and division occurs, may account for the larvae ridding themselves of toxic compounds before pupating (Bowers, 1993). It is unclear why the 1 was not found in the Sh instar of Pyrrhopyge pseudophidias.
39 5. LINKING STATEMENT
The first manuscript investigated the link between three cancer-active compounds and the insects that feed upon the plant from which these compounds were isolated. The active compounds had already been isolated from the plant. An interesting component of the work was the comparison between life stages of each insect, which was possible because the insects were raised in captivity. This was not possible in the second study.
In the second manuscript, the plant-insect association was investigated from an earlier stage of the drug-discovery process. The plant was chosen specifically for bioassay-guided fractionation because it was associated with severallarge, aposematic beetles. The interesting ecological relationship between the insect and its host plant was used as a potential indicator of pharmaceutical value. As weIl, the plant was chosen specifically to enable the investigation of the differences between treatment of host plant compounds by generalist and specialist beetles species from the same family. In the second study, tre active component of the plant was isolated by the first author through bioassay-guided fractionation of the crude extract of the plant.
40 6. MANUSCRIPT 2
A novel sesquiterpene with anti-trypanosomal activity isolated from the plant Mikania guaco and its assimilation by two specialist beetles
ClARA RAUDSEPP-HEARNEY AHMED HUSSEIN? and TODD L. CAPSON1,2*
1Smithsonian Tropical Research Institute Apartado 2072, Balboa, Ancon, Republic of Panama
2Department of Plant Science McGill University 21,111 Lakeshore Road Ste. Anne de Bellevue, Quebec H9X 3V9, Department of Plant Science, McGill University
3Centro de Investigaciones Farmacognosticas de la Flora Panamefia (CIFLORP AN), Facultad de Farmacia, Universidad de Panama, Apartado 10767, Estafeta Universitaria, Panama, Republica of de Panama, University of Panama
*To whom correspondence should be addressed. E-mail: [email protected]
41 ABSTRACT
Bioassay-guided fractionation of the methanolic extracts of the plant Mikania guaco resulted in the isolation of the novel sesquiterpene lactone Guaconone, which was shown to be active against the amastigotic form of Trypanosoma cruzi, the causative agent ofChagas' disease. Compound 1 was isolated using HPLC and standard chromatographie techniques and the structure was elucidated using 2-D NMR techniques and mass spectrometry. Three coleopteran species (Chrysomelidae) that use Mikania guaco as a host plant were investigated for their ability to assimilate 1. The aposematic specialists Eugenysa coscaroni and Echoma anaglyptoides sequester 1 while the visually duller generalist Platyphora ligata does not contain measurable quantities of the compound. The fact that two aposematic specialist insect species are sequestering a pharmaceutically active compound suggests that these two criteria could be useful for selecting plants with compounds that have anti trypanosomal activity.
Key Words - Mikania guaco, Eugenysa coscaroni, Echoma anaglyptoides. Platyphora ligata, Cole optera, insect-plant interactions, Chagas disease, sequestration.
42 6.1. INTRODUCTION
Tropical insects represent an enormous group of organisms that contain a great diversity of organic compounds (Sittenfeld et al 1999). Many insects selectively sequester secondary metabolites from their host plants for uses such as defence, mating and colouration (Harborne, 1999; Geuder et al., 1997). These defensive compounds are similar in structure to the molecules that are responsible for most pharmacological activity in plants (Sittenfeld et al 1999). It has been suggested that insects that assimilate toxic compounds may provide compounds with pharmaceutical properties; however the relatively small amount of crude extract available to pursue bioassay-guided fractionation has been an impediment to research
(Sittenfeld et al., 1999). As part of the activities of the Panama International
Cooperative Biodiversity Groups pro gram (Mendoza et al., 2003), we have used aposematic insects -those which advertise the presence of toxic compounds with bright colouration- as guides to plants with biologically active compounds.
Mikania (Asteraceae) is the largest genus of tropicallianas, representing over
300 species. It is quite common in the national parks surrounding Panama City, in the Republic of Panama. Mikania guaco is a thornless, shrubby vine reaching about 2 m in height growing in low and mid-elevation tropical forests (Darcy 1987). They produce wide, bright green, heart-shaped leaves and white inflorescences over the mid- and late-wet season (July through November) (Windsor et al., 1995). The underside of the leaf has a distinctive bright purple colouration.
Mikania spp. have been found active against snakebites, skin afflictions, fever bronchitis, rheumatism, asthmas, sore throat and cancer (Celeghini et al. 1999; Paul,
43 2000). The well-known herbaI remedy 'guaco' of Brazilian origin, actually refers to
Mikania glomerata and not Mikania guaco, which has not been studied for its medicinal value. A diterpene from Mikania obtusata was active against the trypomastigote form of the parasite Trypanosoma cruzi, which is responsible for the spread of infection from cell to cell and is also the form that is transmitted to the insect vector (Alves et al., 1995). There is currently no treatment for the intracellular
(amastigote) form of the parasite which is responsible for disease manifestations in hum ans (WHO, 2000; Gelb & Hol, 2002).
While Mikania guaco has not been studied for pharmaceutical propert~s, the plant has yielded germacranolides and sesquiterpene lactones (Limberger, 1998;
Rungeler et al., 2001).
Regular monitoring of populations of Mikania guaco in two national parks in the Republic of Panama yielded three insect species feeding on this species in sufficient quantities for analysis.
1. Eugenysa coscaroni Viana is the largest Cassidine beetle in Panama with a body
length of 16-22 mm (Windsor & Choe 1994). Very little is known about its
ecology but it is very visible when feeding, long-lived, raises live young and is an
extreme specialist on Mikania guaco. This beetle is strikingly coloured, with
bright red and black elytra and can be spotted from a distance feeding on its host
plant.
2. Echoma anaglyptoides is another Cassidine beetle (Chrysomelidae) that is similar
in appearance to Eugenysa coscaroni. It is also an extreme specialist on Mikania
guaco and has been observed feeding on the flowers of this plant species.
44 Experiments have suggested that both larvae and adults can only feed on flowers
or young leaves of Mikania guaco (Windsor et al 1995). It has brilliant orange
and black elytra that have been hypothesized to signal distastefulness (Windsor et
al 1995).
3. Platyphora ligata Sta1 (formerly Doryphora ligata) is a genera1ist specils that
feeds on Mikania guaco and has a very broad distribution, ranging from Mexico
to Peru. Very little is known about its ecology although it is relatively common in
Panama. Platyphora ligata is a smaller beetle and is not as striking in appearance
as either Eugenysa coscaroni or Echoma anaglyptoides.
The low number of species observed to feed on Mikania guaco combined with the absence of any obvious physical defence against herbivory suggests that the species may be chemically well-defended against insect predation. The insects that use Mikania guaco as a host plant appear only sporadically and it is rare to find plants damaged by herbivory. Aposematic theory dictates that a bright appearance and attention-attracting behaviour may signal the presen:e of defensive chemical compounds within the in sect that are either biosynthesized or sequestered from the host plant (Bowers 1993). Ecological theory on chemically-mediated insect-host plant interactions suggests that the two similar specialist species (Eugenysa coscaroni and Echoma anaglyptoides) are more likely to sequester host plant secondary compounds than the less aposematic generalist species. The theory states that specialist insect species -those that feed on only one or a few plant species-have co evolved with their specifie host plant and have had a greater opportunity to adapt to their host plant's secondary defence compounds and even make use of the se
45 defensive compounds for their own defence (Ehrlich & Raven, 1964). Using biological assays against Trypanosoma cruzi we compared both the specialist and generalist Chrysomelid beetles for their ability to sequester biologically active compounds from Mikania guaco.
a
c d Figure 6.1. (a) Mikania guaco, (b) Echoma anaglyptoides, (c) Platyphora Iigata, (d) Eugenysa coscaroni.
46 6.2. MATERIALS AND METHODS
6.2.1. PLANT AND INSECTS
Plant material. Initial activity against Trypanosoma cruzi was found in plants collected from Altos de Campana National Park in 2002. Crude extracts of Mikania guaco were found to have ICso values of 4f..lg/mL against the amastigote form of T. cruzi (the bioassay is described in section 3.3.2.). Further plant specimens for chemical extraction were collected from the same location. 600 g of fresh young leaves were collected in, yielding a total of 20 g of dried crude extract. Voucher specimens were deposited in the ICBG herbarium at the Smithsonian Tropical
Research Institute (voucher number B3392).
Insect material. Insects were collected from their host plant Mikania guaco within
Altos de Campana National Park and Chagres National Park, both located in the
Republic of Panama. Collections occurred between May and November of 2002
(with the exception of Echoma anaglyptoides which was collected during a
biodiversity inventory initiated by the ICBG in 2000), during which time individual
plants were monitored regularly to check for any insects feeding on them.
Chemical Extraction. Insects were killed by deep-freezing at -80°C and then stored in
methanol at the same temperature. Insects were starved for at least 36 hours prior to
deep-freezing to ensure that the gut was empty. Control experiments were performed in which insect guts were removed prior to extraction ta ensure that plant residue did
not interfere with the analysis of secondary metabolites. Insects and plants were
47 homogenized with 5ml of cold methanol for 5 minutes with a Waring blender foUowed by treatment with a Polytron homogenizer (Brinkman Instruments, Inc.) at
O°C for two minutes. The mixture was filtered under vacuum and the marc was then washed with 50 ml of methanol. The extract was concentrated by rotary evaporation, lyophilized, and stored at -80 oC until further use.
6.2.2. BIOASSAy-GUIDED FRACTIONATION OF MIKANIA GUACO
Compound Isolation. Analysis of the crude plant extract by TLC and HPLC showed
the presence of tannins. The extract was detannified by filtering the crude extract
through 15 g of polyamide and 5 g of silica by flash chromatography (Still et al.,
1978).
The active component of Mikania guaco was isolated by HPLC using
bioassay-guided fractionation. AH samples were subjected to filtration through
reverse phase octadecyl-functionalized silica gel before HPLC analysis. The HPLC
system was from Waters (Milford, Massachusetts) equipped with a photodiode array
detector (Waters, Milford, Massachusetts). HPLC analyses employed a C18 Nova
Pak column [analytical: 4.6 X 250 mm, 4 micron pore size; preparative: radial
compression module kit, Nova-Pak, two 25 X 200 mm segments, 6 micron pore size],
with linear gradients of CH3CN and H20. Crude extracts were then fractionated
using a Nova-Pak Cl8 preparative column (Waters) (25 X 200 mm, 6 !lm pore size).
AU fractions were sent to the bioassay for the disease which the crude extract showed
48 activity against (described in Appendix A). AIl fractions were tested twice in the bioassay to confrrm activity. Active fractions were then subjected to the same fractionation process (same linear gradients of CH3CN and H20) which was repeated until the active component of the plant was isolated. The active component was subjected to TLC under different conditions to verify that it contained one purified compound, and this was verified again using NMR. The structure of the isolated compound was investigated using NMR and spectroscopic techniques with the help of chemists from the University of Panama and Oregon State University.
Fractions were tested and subjected to additional purification until purified compounds were obtained.
Larger quantities of purified compounds were isolated using standard column chromatographie techniques. 18 g of crude extract were dissolved in ethyl acetate and filtered by vacuum through a column of 500g compacted silica. Crude extract was supported on silica gel 60 (0.063 - 2 mm) and dried for 0.5 hours. Fractions obtained using this technique were compared by TLC to fractions isolated by HPLC and the active component was easily identified and isolated using a series of column separations. These larger quantities were used for structural elucidation by NMR and
Mass Spectrometry (performed by second author Ahmed Hussein).
49 6.2.3. INSECT-PLANT CHEMICAL-EcOLOGY
Insect extracts were filtered through reverse phase octadecyl-functionalized silica gel prior to analysis by HPLC. Extracts were analyzed using the same instruments and conditions used for plant extract analysis.
Compounds isolated from insects were initially compared by retention times and UV spectra obtained from HPLC analysis to the purified compounds isolated from Mikania guaco. The identity of insect-derived 1 was confirmed by mass spectrometry.
6.3. RESULTS
6.3.1. COMPOUND ISOLATION AND ELUCIDATION OF STRUCTURE
Following anti-trypanosomal activity, the novel sesquiterpene lactone 1 was isolated from Mikania guaco and shown to have an activity of 9 llg/mL in the intra cellular T. cruzi bioassay. The name Guaconone is proposed for compound 1 (Figure
6.2.).
50 Figure 6.2. Structure of novel sesquiterpene lactone Guaconone, isolated from Mikania guaco. -( o
OH J=o
6.3.2. INSECT-HoST PLANT CHEMICAL ECOLOGY
Compound 1 was found to be sequestered by both Eugenysa coscaroni and
Echoma anaglyptoides while the extract of Platyphora ligata did not contain a trace
of the compound (Figure 6.3.). The identity of 1 isolated from Eugenysa coscaroni
and Echoma anaglyptoides was confirmed by mass spectrometry. The active
compound Guaconone was found to be sequestered by two of the three insects in the
study.
51 Figure 6.3. HPLC results comparing retentions times of the pure compound Guaconone, crude extract of plant Mikania guaco, and extracts of three beetles that feed on the plant.
-{ Guaconone o o
5.00 10.00 15,00 20,00 25.00 30.00 35.00 l, .. """ ..Minutes :J .. .J ~o
Crude extract Mikania guaco 1LCJLJ10.00 20.00 30.00 4000 5000
Platyphora ligata
Echoma anaglyptoides '!l1000 :LJ2000 30.00 4000 50.00
Eugenysa coscaroni
52 6.4. DISCUSSION
Specialist insect species are considered more likely to sequester toxic host plant compounds than generalist species since specialist predator(s) are more likely to develop a tolerance to defensive secondary metabolites in a plant through co evolution than generalist insect species (Ehrlich & Raven, 1964). Thus the presence of Guaconone in the specialist insects may be related to their enhanced ability to tolerate Guaconone and may also be related to the sequestration of the compound in order to deter predators of the beetles.
HPLC analysis of the detannified MeOH extracts ofboth Echoma
anaglyptoides and Eugenysa coscaroni revealed a set of extremely polar compounds
and a c1uster of compounds of mid-range polarity, many of which correspond with
compounds from the crude extract of Mikania guaco. This finding suggests that
many of the compounds found in the insects originated in the host plant. The
isolation of Guaconone resulted from bioassay guided fractionation for anti
trypanosomal activity; however it is highly likely that additional biologically active
compounds are also present Mikania guaco.
The broader idea behind this study was to investigate whether aposematic
insects could be used to guide natural products chemists to plants with interesting
chemical compounds. The definition of aposematism links an unpleasant or toxic
quality of a prey (unpalatibility) with an advertisement of this feature (waming
colouration) (Bowers, 1993). Unpalatable insects are thought to advertise their
defence by conspicuous colouration, gregariousness and sedentary behaviour
53 (Bowers, 1993). AIl three insect species in this study were bright and apparent to the human eye, although the generalist species Platyphora ligata was somewhat less so.
Platyphora species have been shown to sequester plant compounds for defensive secretions (saponins, pyrrolizidine alkaloids), but in this case the species is obviously not using the same mechanism of defence as the two specialist species (Pasteels et al.,
2001).
It was interesting to note that the two specialist species were extreme specialists on the young leaves oftheir host plant (and only the young leaves were active in tre T. cruzi bioassay) and were similar in appearance to each other. Both were brightly coloured and exhibited behaviour that made them visible and vulnerable to predators.
As many criteria as possible should be used to determine whether an insect is advertising plant-derived defences. It would appear also that specialist vs. generalist lifestyle might be as useful a criteria for indicating medicinaIly-interesting compounds as aposematism because the generalist species (Platyphora !igata) that did not sequester Guacanone was also somewhat aposematic. Aposematism is not clearly defined and there is recognition from the literature that it is a relative term and depends largely on how predators see and interpret visual cues from their prey. More studies ccmparing the treatment of host plant secondary compounds by specialist and generalist predators would show if it is strategicaIly useful to distinguish between these two groups in the context of drug disco very.
54 7. GENERAL DISCUSSION & CONCLUSIONS
The results from this work suggest that there is potential for using insects as guides in the drug discovery process. Two other plant-insect associations were investigated during the course ofthis master's project but more work is needed to investigate the relationship between active partitions of the plants and their associated insects. The work that was initiated with the plant Hasseltiafloribunda and Mabea occidentalis could be followed up by the next graduate student who will be continuing in this specifie line of investigation. Before any conclusions can be drawn as to the usefulness of using insects as guides to medicinal compounds, a larger body of work dealing with the subject will be required.
Although it seems that aposematic insects could potentially indicate active plant compounds useful to medicine, it is unclear which criteria are most important for indicating interesting and active chemical compounds. Is it more important for the insect to be aposematic or an extreme specialist on its host plant? It is important to recognize that what is aposematic may differ between different predators. Insect, bird and mammals predators locate and identify prey using different senses and methods (Keeton, 1967). As well, unpalatable species may be mimicked by palatable species, and the presence of varying proportions of palatable look-alikes could affect the efficacy of aposematism. There is a limit to how useful insects will be in the field of medicine due to these uncertainties.
While a broad survey of the chemical-ecological relationships between insects and their host plants is needed, there is also a need for more in-depth studies of several insect-plant associations in order to determine chemical pathways and how
55 chemical compounds affect the behaviour of insects and their predators. And finally these studies must be linked to compounds of medicinal value. A weakness in this thesis is the lack of bioassays testing whether medicinally-active compounds found to have been sequestered by certain insects were being sequestered specifically for defensive purposes. U sing aposematism as the main criterion for insects to be used as guides to new medicines assumes that the insect is preferentially making use of the plant compounds that are active in our specifie disease bioassays for defensive purposes and that it is these compounds that the insect is advertising with bright colouration. A longer study requiring the successful breeding of captive insect colonies would inc1ude bioassays with preœtors to test whether the plant compounds active against human disease affect predation rates on the insects that sequester these compounds from their host plants (Theodoratus & Bowers, 1999).
There are many other interesting avenues of investigation tmt could be pursued in relation to the question of how insects could be used in bioprospecting.
Sorne studies have shown that plants can release toxins as a reaction to herbivory
(Edwards & Wratten, 1985; Baldwin, 1988). It would be interesting to test plant activity in disease bioassays in the presence and absence of their herbivore. This line of investigation could lead to new compounds that are not usually present in plants in the absence of their herbivores.
Another avenue of investigation that would link insect-plant chemical ecology to the pharmaceutical industry is the question of how insects transform plant compounds. In many cases, insects modify their host plant's secondary compounds
(e.g. esterification of a pyrrolizidine alkaloid, glucosylation of a flavonoid)
56 (Hartmann et al., 1990; Wiesen et al., 1994). AIso, the way that insects detoxify chemicals using enzymes could be used to aHow the use of otherwise overly toxic chemicals as human drugs (Schroder et al., 1998; Nitao, 1989).
In order to be able to use insects as efficient and worthwhile guides to novel medicines, a set of criteria would have to be developed to direct the search. This study suggests that there is sorne overIap between the secondary plant compounds that insects sequester and the compounds that are potentiaHy of interest to medicine.
However not aH colourful insects willlead us to new medicines.
The difference in defensive strategies between life stages of an insect is more of interest to ecology than medicine (Bowers & Collinge, 1992). For example, it would not be feasible to generalize that the 4h instars of aH caterpillars are the instars that will indicate medicinal activity in a plant. However it is important to ascertain whether larvae or adults are more accurate indicators of interesting chemical compounds. For examp1e, l wasunable to investigate how the larval stages of
Eugenysa coscaroni, Echoma anaglyptoides and Platyphora ligata treat the compound Guaconone in their host plant Mikania guaco. The adult stage of
Platyphora ligata (which does not contain the compound Guaconone) is less
aposematic than the adult stages of Eugenysa coscaroni and Echoma anaglyptoides
(both of which sequester Guaconone), but possibly a different trend would appear if
one was to compare the larval stages. Non-aposematic adults should not be ignored
as often the larvae of these adults are aposematic and could be signalling chemical
defence. In the case of Vismia baccifera the adults of both moth species inc1uded in
the study were non-aposematic and their extracts did not contain measurable
57 quantities of defensive compounds. In this case, it was crucial to look at the larval stages.
Another criterion that could be useful is whether an insect is a specialist on its host plant or a generalist. In both papers, specialist and generalist insect species were investigated to see whether they treated toxic chemical compounds differently. In the case of Vismia baccifera, the generalist and specialist species treated the cancer active compounds in the same way. This has been reported several times in the literature. In a paper by Szentesi and Wink (1991), an aphid species and a bruchid
species that both fed on at least 6 other plants, their parasites, and one specialist beetle species, were al! able to sequester toxic quinolizidine alkaloids from the same
host plant. However in most cases, specialist insects are better able to assimilate the
secondary compounds of any particular plant (Bowers, 1993). This was shown in the
study on Mikania guaco and its insect predators.
Nearly aU plants, irrespective of the chemical or mechanical defence system
they have evolved, possess a few pathogens or predators. But compared to the
number of potential enemies, the number of specialized pest species is exceedingly
small (Wink & Romer, 1986). Specia1ists that have overcome a plant's defence
system do not contradict the theory that secondary metabolites are important for the
fitness of a plant. They show that natural chemical defence is not absolute and that
resistant pathogens evolve, as is the situation with marrmade chemicals employed for
plant protection. In fact, an 'arms race' between a specialized insect and a plant has
the potential to produce complicated and novel chemical structures over an
evolutionary time period (Ehrlich and Raven, 1964). For medicinal use, l would
58 propose that specialist insects could serve as the most useful indicators of interesting and highly active novel chemical structures. Future research in this field could investigate this theory further.
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66 APPENDIX A: BIOASSAYS
1. CANCERBIOASSAY
The protocol for the anticancer assay was developed by the National Cancer
Institute (NCI). The ICBG uses this assay with the modification that instead ofusing a panel of 60 celllines, 3 celllines are used: breast tissue (MCF-7), lung (H-460) and central nervous system (SF-268). These lines were chosen for use in a deve10ping country as they cover a broad spectrum of cancer types and are relatively easy to culture.
The protocols used to culture celllines and to measure cytotoxicity are identical to those used by the NCI, with the exception of the use of XTT instead of sulforhodamine B to measure viability. For the testing of crude extracts, after dissolving in DMSO, the extracts are initially tested at a single concentration of 100
~glmL. Extracts that cause at least 30% cytotoxicity are then tested at five concentrations (1.0, 3.2,10.0, 31.6 and 100 ~g/mL), the data from which are used to calculate GIso values. Extracts that demonstrate GIso values of 10 Ilg/mL or less are considered active.
2. TRYPANOSOMA CRUZIBIOASSAY
The ICBG laboratory employs a recombinant strain of Trypanosoma cruzi that expresses the Escherichia coli l3-galactosidase gene to test candidate antitrypanosomal compounds (Buckner et al. 1996). Transfected parasites catalyze a colourimetric reaction with chlorophenol red-I3-D-galactopyranoside (CPRG) as
67 substrate and the amount of l3-galactosidase activity is directly proportional to the number of transfected parasites. The assay is performed in a 96-weIl plate and is analyzed with a microplate reader.
The ICBG has recently developed a bioassay with the intraceIlular
(amastigote) form ofT. cruzi, which was also used during this study. The trypomastigote stage of T. cruzi is responsible for spreading the infection from ceIl to ceIl. It is short-lived and rapidly transforms into the amastigote form upon entering into the host-ceIl cytoplasm. It is the amastigote form of the parasite which is the most relevant clinically. The bioassay uses human fibroblasts as host-cells to betler mimic natural conditions. The trypomastigotes are allowed to invade the host cell mono layer ovemight prior to exposing the infected ceIls to the test substance.
Controls are performed to determine the effect of the test compound on both the parasite and the host-cell. This is useful when examining the specificity of activity of test substances. Plant extracts are initially tested in duplicate at two concentrations,
50 and 10 j..Lg/mL, and extracts that show ICso values lower than 50 j..Lg/mL are considered active. Extracts that are active at a single concentration are reconfirmed by testing in duplicate at 50, 10, and 2 j..Lg/mL, and the same concentrations are used for testing fractions from bioassay-guided fractionation.
68