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Screening of biflavonoid compounds and British Columbian bryophytes for antiviral activity against potato… Hillhouse, Brian Jason 2003

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Screening of Biflavonoid Compounds and British Columbian Bryophyt for Antiviral Activity Against Potato Virus X by BRIAN JASON HILLHOUSE B.Sc. (hons.), The University of British Columbia, 2000  A thesis submitted in partial fulfillment of the requirements for the degree of  M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Botany) We accept this thesis as conforming to the required standard  The University of British Columbia April 2003 © Brian Jason Hillhouse, 2003  In presenting  this  degree at the  thesis  in  partial fulfilment  of  University of  British Columbia,  I agree  freely available for reference copying  of  department  this or  publication of  and study.  his  or  her  representatives.  may be It  is  requirements that the  for  an  Department The University of British Columbia Vancouver, Canada  advanced  Library shall make it  that permission for extensive granted  by the  understood  head  that  this thesis for financial gain shall not be allowed without  permission.  DE-6 (2/88)  I further agree  thesis for scholarly purposes by  the  of  my  copying  or  my written  Abstract Plant viruses are responsible for causing significant losses to the agricultural industry. Recent investigations of antiviral compounds have suggested that biflavonoids may be a group of compounds which cause powerful inhibition to a broad spectrum of viral pathogens.  Due to the high content of biflavonoids reported in bryophytes, this  project has screened 50 bryophyte samples which represent 41 species for Potato Virus X ( P V X ) inhibition using a local lesion assay in Chenopodium quinoa. As a result of this screening, bryophytes have been identified as a prominent antiviral group; 29 of the 41 species were shown to cause greater than 75% inhibition when tested at 9.1mg/mL. In addition to screening bryophyte extracts, several biflavonoid compounds were tested directly. When tested at 1.38mg/mL robustaflavone and hinokiflavone where shown to be highly antiviral and exhibited P V X inhibitions of 85% and 78% respectively. Amentoflavone-7,4',4'trimethyl ether showed a marginal P V X inhibition of 29% and amentoflavone did not inhibit P V X at this concentration. Several compounds from the Columbian medicinal plant Iryanthera megistophylla have also been screened for inhibition of P V X . O f these compounds, iryanterin K , cinchonain lb, cinchonain la, procyanidin B-2 and cinchonain Ha have been shown to be highly active against P V X when tested at 9.1 mg/mL.  Table of Contents Abstract List of Tables List of Figures Acknowledgements 1 Introduction 1.1 Viruses 1.2 Significance of Plant Viruses 1.3 Methods of Controlling Plant Viruses 1.4 Potato Virus X 1.5 The Importance of Potato Crops 1.6 Flavonoids 1.7 Biflavonoids 1.8 Mechanism of Action of Biflavonoids 1.9 Bryophytes 1.10 Objectives 2 Methods 2.1 2.2 2.3 2.4 2.5  Collection and Identification of Bryophytes Extraction of Bryophytes The Antiviral Assay Biflavonoid Testing Fractionation of the Bryophyte Hylocomium splendens  3 Results & Discussion 3.1 Anti-viral Activity of Bryophytes 3.1.1 Results were not obtained from all samples collected 3.1.2 The majority of species caused a high level of inhibition. 3.1.3 The correlation between activity and error 3.1.4 High error is a result o f inconsistent infection production 3.1.5 Eight species with multiple samples 3.1.6 Relatedness among bryophyte samples 3.2 Anti-viral Activity of Biflavonoids 3.3 The Chemistry of Hylocomium splendens 3.4 Anti-viral Compounds from Iryanthera megistophylla 3.5 Summary 4 References  1 1  List of Tables Table 1: Some unique bryophyte biflavonoids  18  Table 2: Bryophyte collection  21  Table 3: The anti-PVX activity of tested bryophyte species  31  Table 4: The local lesion assay results of sample 127 H. splendens  35  Table 5: The anti-PVX activity of the biflavonoid compounds  41  Table 6: A n t i - P V X activity of Iryanthera megistophylla compounds  50  iv  List of Figures  Figure 1: The basic flavonoid unit  11  Figure 2: Summary of flavonoid synthesis  12  Figure 3: 5,7,4'-trihydroxy-8-methoxyflavone  13  Figure 4: Quercetin  13  Figure 5: Five week old Chenopodium quinoa  26  Figure 6: Chenopodium quinoa leaf showing lesions  27  Figure 7: Relationship between inhibition and uncertainty in bryophyte samples  33  Figure 8: The phylogeny of the bryophyte samples tested  39  Figure 9: Robustaflavone  44  Figure 10: Hinokiflavone  44  Figure 11: Amentoflavone  44  Figure 12: Amentoflavone-7,4',4'trimethyl ether  44  Figure 13: Comparison of the hexane and ethyl acetate fractions of H. splendens  46  Figure 14: P V X inhibition caused by H. splendens fractions  '.  47  Figure 15: 5',3"'-Dihydroxyrobustaflavone  48  Figure 16: Megislignan  52  Figure 17: Procyanidin B-2  52  Figure 18: Megislactone  52  Figure 19: Cinchonain  52  Figure 20: Grandinolide  52  Figure 21: Cinchonain Ha  52  Figure 22: Iryantherin  52  v  Acknowledgements I would like to acknowledge the following people who have assisted and supported this project. I am very grateful to Dr. Neil Towers for his mentorship and for making this project possible. I also wish to thank Dr. Chris French for his guidance and funding in this project. Thanks to Dr. Dong Sheng Ming for his friendship, and many lessons in chemistry. Dr. Dong Sheng M i n g also played a critical role in the isolation of the Iryanthera and Hylocomium compounds. Thanks to Mike Boutihillier for providing generous instruction and assistance. Thanks to Olivia Lee, Dr. W i l f Schofield and Shona Ellis for assisting me in my study of bryology and sharing their enthusiasm in this area. Funding of this project was made possible with contributions from Agriculture and Agri-Food Canada, PhytoDiagnostics Company Ltd. and N S E R C . I would also like to thank the many members of Towers lab including: Keith Pardee, Dr. Eduardo Jovel, Dr. Andres Lopez and Dr. Kevin Usher. Their friendship, support and guidance made this project a valuable experience. Thanks to my wife, Beth Hillhouse for her editing assistance and her unconditional support throughout this project. Thanks also to my parents for their assistance and encouragement.  vi  This thesis is dedicated to my parents, Dave and Marguerite Hillhouse.  1 Introduction 1.1 Viruses Viruses are quite unlike any other pathogen. The question of whether viruses are living organisms is still frequently debated. Viruses are not capable o f motion, nor do they grow or consume nutrients for energy. In fact, viruses are very complex chemical molecules which depend entirely on host cell machinery to reproduce. Webster's New World Dictionary defines a virus as: "Any of a kingdom (Virus) ofprokaryotes, usually ultramicroscopic, that consist of nucleic acid, either RNA or DNA, within a case of protein: they infect animals, plants, and bacteria and can reproduce only within living cells so that they are considered as being either living organisms or inert chemicals. " -Webster's New World Dictionary, 1998. In comparison with other pathogens, viruses are unique in that their components are intermixed with the cellular components of their host and thus the two organisms are virtually indistinguishable. This intermixed relationship makes it very difficult to eradicate the virus without harming the host.  1.2 Significance of Plant Viruses Plant diseases cause significant losses to the agricultural industry. It has been estimated that total agricultural losses due to plant disease are as high as US$60 billion per year (Klausner 1987). Fungi and viruses are responsible for the majority of these losses and viruses are reported as being the second most damaging plant pathogen (Mathews, 1991). Currently, viral diseases are a problem for most agricultural crops, but the extent o f damage caused by viral infections can vary greatly. Viral infections  1  typically result in a substantial decrease in crop yield and/or decrease in the quality of a product. Once a crop is infected with a virus there is no means of removing or exterminating that virus from the crop. Because of this, viral infections in fruit trees and other high investment perennial crops can be extremely costly. In addition to the cost o f replacing crops, virus infections reduce plant productivity, substantially decreasing crop yields and grower revenues. Some of the more benign viruses are known to cause yield decreases of 25% or more. Some of the most affected crops include potato, tobacco, tomato, cucumber, melons, strawberry, cabbage and radish (Tokuda et al. 1981). There is great demand for methods to reduce the damage caused by viruses and controlling viral diseases. Viral infections in cultivated plants are extremely common. A n example of the abundance of viral infection has been shown through a survey of the popular orchid genus, Oncidium. Upon examination of this genus, investigators were unable to find any virus-free specimens (Chia et al. 1991). Once virus-free orchids were established, it was shown that removal of viruses yielded a 65% increase in inflorescence size and a 21% increase in photosynthetic capacity (Chia et al. 1999). Other crops, such as onion and garlic, are also known to be universally infected with viral pathogens. A l l forms of commercially produced garlic worldwide have been shown to be infected with the onion yellow dwarf virus (Rice et al. 1998). In addition to directly reducing crop yields and damaging plants, viruses can also weaken plants and make them more susceptible to damage caused by other factors. Virus infected plants are more easily attacked by fungal, bacterial and even other viral diseases. Plants that are already weakened by a viral infection are less able to cope with the  2  secondary infection and the result can be devastating. When two different viruses infect the same host at the same time, the result is known as "plant viral synergism". In this situation the plant may display severe disease symptoms and i f the plant survives, the observed damage may be greater than the combined damage (loss of yield) expected from the two separate viruses (Pruss et al. 1997). With over 23 viruses that have been reported to cause damage to potato alone (Harvath, 1967), viral synergism can easily occur and when it does, it can render the crop unmarketable. The ability of plant viruses to be transmitted effectively to other plants causes them to be extremely problematic to plant growers. Currently, the only strategy to reduce the impact of plant viruses is to sow viral free material. Unfortunately, virus-free seed can be very expensive and only provides temporary relief, as viruses are well adapted to re-infect these fields. It is surprising how successful these simple organisms are at dispersing themselves. Some viruses are capable of transmission through various kinds o f mechanical disturbances that bring an infected plant or its fluids in contact with another plant. Many viruses take advantage of insects as vectors to spread to other plants; for example the Potato Leaf-Roll Virus is highly adapted to using insects as vectors for dispersal. The Potato Leaf-Roll Virus is capable of proliferating in various aphid species and thus once the virus is present in the insect, it is capable of infecting every susceptible plant that the insect comes in contact with. This highly effective form of dispersal makes it very difficult to keep viruses from entering crops. Insects also make excellent vectors as they often place the viruses directly into the phloem of the plant and thus the virus is capable of rapidly spreading throughout the plant.  3  Fungi are also significant vectors o f viruses. In this case, damage may be caused by both the fungus and the virus. The four most significant fungal vectors are Olpidium brassicae, Plymyxa graminis, Spongospora subterranea and the controversial potato wart fungus (Synchytrium edobioticum) which was recently found in Prince Edward Island potatoes (Teakle, 1969). Viruses persist inside their fungal hosts and may be transmitted to crops which the fungal host infects or comes into contact with.  1.3 Methods o f Controlling Plant Viruses Unfortunately, there is are few effective methods to prevent or treat viral infections in plants. The most common methods of controlling plant viruses are passive, which do not attempt to directly remove or inhibit viruses. These strategies include: removing infected plants, using insecticides to control vectors, planting barrier crops and protecting crops from damage caused by machinery and the environment. The most effective method of viral control which is available to today's growers is the planting of certified virus-free seed. It is only recently that virus-free seed for most crops has been accessible to growers. Initially, virus-free seed was produced through breeding practices, which simply selected for the most productive plants. Twenty years ago, we still had no means of removing the more persistent viruses such as Potato Virus X ( P V X ) in potato. A s a result of this, until recently, P V X was endemic to all potato growing operations (Klein & Livingston, 1983). Early methods of producing virus-free seed material took advantage of the discovery that viral concentrations are lower in immature leaves and often absent from the growing meristem (Limasset et al. 1949). Thus, in the 50's and 60's, virus-free plants  4  were produced by growing plants from excised meristems. However, this method was slow and only effective with some viruses. Another early technique for eliminating viruses from propagative material was through the use of heat treatments. Prolonged exposure to warm growing conditions has resulted in the elimination o f several viruses. Potato leaf-roll virus was first eradicated from potato tubes by incubating tubers in air at 37.5°C for at least 25 days (Kassanis, 1949). P V X can be removed from potato by incubating shoots at 37°C for 15 weeks (Mellor and Stace-Smith, 1967); however thermotherapeutic methods are limited as they can require long incubation times and plants already weakened by the virus infection can often be destroyed in the process. A combination of heat therapy and meristem culturing has resulted in much higher success. But even then only a fraction of the subjected plant material is actually freed of its viral contaminants. For example, the success rate for producing virus-free potato plants infected with P V X is only 4% of the excised plant samples which were subjected to a 23 day treatment (MacDonald, 1973). The most effective methods of virus elimination involve the use of chemotherapies or antiphytoviral drugs in combination with meristem removal and thermotherapeutic methods. The first available phytoviral drug was dimethyl sulfoxide which was made available in the late 60's (Herschler and Wash, 1967). Although dimethyl sulfoxide is capable of inhibiting viral reproduction and growth, because of its high toxicity it has been of little practical use to the agricultural industry. The nucleoside analogue ribavirin (Virazole™) is the most commonly used antiphytoviral drug. Ribavirin was one of the first drugs approved for use in humans for the  5  treatment of virus diseases. The ability of ribavirin to inhibit plant viruses was first discovered in 1976 (Schuster, 1967) and then further investigated in 1977 (Lerch, 1977) and ribavirin was quickly used for the production of virus-free tissue cultures (Hansen 1979, Klein and Livingston 1982, Cassells and Long 1982). Over the years, ribavirin has been shown to be very useful in the virus elimination from plants. Applying a ribavirin and heat treatment to cultured meristems can increase the incidence of formation of virusfree plants and thus decreases the time and cost of indexing treated plants (Hansen, 1984). Phytodiagnostics Company Ltd. is a private biotech company based out of Sidney, B C which removes viruses from plant material used by seed potato growers. Researchers of this company have found that treating tissue cultured meristems with high levels of heat and ribavirin is the most versatile and useful means of deriving virus-free propagative material (Ellis, 2001). Once the tissue culture is treated, the plant is rejuvenated and tested for the presence of viruses by means of E L I S A (enzyme-lined immunosorbent assay). Once the above methods have been used to produce virus-free plants, tissue culture methods are used to rapidly clone seed plants. There is still a great demand for a method of directly preventing or eradicating viral infections from plants growing in fields. Dimethyl sulfoxide was also one of the first substances patented and marketed for use as a protective spray against viruses in field crops (Herschler, 1967). This method quickly proved to be impractical as complete coverage of the plant is needed in order to provide protection and the effects of this compound are very short lived (Mikami et al. 1993). In 1985, a substance known as "The Marvel of Peru" was patented as being able to provide plants with systemic protection  6  from viruses. Unfortunately, this substance is a protein which'is extremely expensive to make and in vivo production has not been approved for agricultural use (Mikami et al. 1993). Recently, fungi in the genus Fomes have been shown to produce large amounts o f 'acidic high molecular polysaccharides' which have systemic antiviral properties (Mikami et al. 1993). Japan Tobacco Inc. and the Institute of Fermentation Organization have developed a strain of the fungus Fomes fomentarius known as JTS 3046 which has a remarkably high production of these compounds. When the culture filtrate is applied to plants, plants were shown to acquire systemic protection against T M V (Mikami et al. 1993). The only shortcoming of these fungal compounds is that they are specific to the Tobacco Mosaic Virus and apparently not effective on other plant viruses. A n area of plant virology which has seen a lot of recent advances is genetically engineered viral resistance. Despite its initial cost to growers, the use of resistant crops is generally one of the most economical and practical methods of controlling plant viruses, as it requires less protective measures and crops sustain less damage.  Resistant crops do  not require the controversial application of herbicides, pesticides, or virucides to eliminate weed hosts, insect vectors, or inoculated viruses. Thus, host resistance is one of the most environmentally safe and durable methods for controlling plant diseases. In many plant-virus interactions, resistance is not easily available or cannot be obtained using traditional plant breeding strategies. Recent advances have shown that engineering plants to express viral coat proteins can provide resistance to the introduced virus (Powell-Abel et al. 1986, Stubbs and Culver 1998, Watanabe et al. 1995). B y inserting proteins which are common to several plant viruses, Watanabe et al. engineered a tobacco  7  plant which possessed resistance to Tomato Mosaic Virus, Cucumber Mosaic Virus and Potato Virus Y (Watanabe et al. 1995). This high level form of resistance which is caused through transgenic expression of viral proteins is known as 'coat protein-mediated resistance', and has been used to produce effective resistance against Potato Virus X , Potato Virus Y , Potato Virus S, Potato Leaf-Roll Virus and Potato Mop-Top Virus in selected varieties (Spillane et al. 1998). Despite the effectiveness of this method and the damage caused by plant viruses, there is considerable consumer opposition to the use of genetically modified crops.  1.4 Potato Virus X This project focused on finding natural chemicals which can inhibit potato virus X ( P V X ) . P V X is a member of the potexviruses, a group of filamentous viruses with positively wound single stranded R N A genomes. In addition to P V X , this group of viruses contains some other very damaging viruses such as cymbidium mosaic virus, which is a significant virus affecting orchids (Agrios, 1997). P V X is the most widespread of all the potato viruses. It is capable of reducing crop yields by up to 20% before visible symptoms appear and ultimately P V X can reduce the crop yield by as much as 50% (Achultz and Bonde , 1944). Many strains of P V X exist and the virus is known to cause systemic infection in crops within the Solonaceae (including tomatoes, peppers and potatoes) and Cruciferae (including the turnip), and can cause local lesions in members of the Chenopodiaceae (including spinach and beets), Amaranthaceae (including the amaranth), and some legumes.  8  The agricultural impact currently caused by P V X in potato is not as severe as that of other viruses such as Blueberry Scorch Virus in blueberry or Plum Pox Virus in tree fruit. Damage caused by P V X has been minimized by the use of seed certification programs. P V X was chosen for use in this study because it is a stable virus which is relatively easy to purify and use experimentally with tests on infectivity. In addition, the consequences of P V X being accidentally released into the environment would not be catastrophic. Due to the nature of viral inhibitors, it is possible that any compounds which are found to be effective inhibitors of P V X will also have inhibitory properties against other plant and animal viruses. This broad spectrum tendency of some antiviral substances makes the use of anti-plant viral assays an extremely cost effective and ethical means of studying antiviral compounds.  1.5 The Importance of Potato Crops Most of the damage caused by P V X affects potatoes; a crop which suffers from heavy pathogen damage. Not only is potato susceptible to more than a hundred pathogens including, fungi, bacteria, nematodes and mycoplasmas, but it is also susceptible to as many as 23 viruses. Damage caused to the potato by late blight pathogen (Phytophthora infestans) in Ireland resulted in the death of 250,000 people in 1845. Currently, diseases affecting this crop are responsible for the hunger of millions living in developing countries. The potato crop is a very important staple, only surpassed in importance by wheat, corn and rice. Although potato crops are presently grown only in temperate areas, cultivation is about to dramatically increase with the introduction of  9  new tropical varieties that are designed to meet the needs of developing countries in warmer climates. The potato is also o f great local importance. The B C Vegetable Marketing Commission lists the potato as the most important vegetable crop in the province. In view of this, we should attempt to increase our potato yields so that we are able to support more of our domestic consumption. The Canadian potato industry is in need of assistance. Due to the recent emergence of the Potato Wart fungal parasite (Synchytrium endobioticum) in Prince Edward Island, Canada was unable to export its potato production to the Eastern United States. We need to invest in the development of cures for potato parasites if we are to keep our potato industry healthy. It has been known for some time that the presence o f viruses, no matter how benign they may seem, can lower crop yields and promote occurrence of other disease. Having viral free crops will not only improve our yields, but also promote resistance to contaminants such as the potato wart fungus which was discovered in P E L  10  1.6 Flavonoids Flavonoids are a type of secondary metabolite common to all "higher plants" (Koes et al. 1994), and are well known for their antiviral properties against animal viruses (Pietta, 2000, Kaul et al. 1985). Flavonoids are characterized as phenolic compounds containing two aromatic rings connected by a three carbon bridge. These compounds are synthesized in plants via the shikimic and malonic acid pathway and are based on the precursor phenylalanine. A simplified depiction of flavonoid synthesis is displayed in Figure 2. Because flavonoids occur almost universally in green land-plants and are not present in algae, flavonoids are thought to have played a fundamental role in the colonization of land habitats (Geiger et al. 1997). Current theory is that flavonoids function to protect the plant from damage caused by U V radiation. 3'  Figure 1: The basic flavonoid unit. The aromatic rings are labeled with red letters and the positions are numbered. Carbons 2,3 and 4 form a three carbon bridge between the two aromatic rings. These carbons may be arranged to form a third ring structure, or left open to form a group of flavonoids known as chalcone.  Several projects have screened flavonoids for activity against a wide range of animal viruses and currently a large number of flavonoids have been shown to possess antiviral properties. A relatively early flavonoid study by Nagai et al. examined 103 different monomeric flavonoids for activity against Influenza A. Their comparison of  11  different flavonoid structures found that no correlation could be made between certain structures and anti-viral activity, but they did find several flavonoids which demonstrated potent antiviral activity.  OH  Or  Chalcone Figure 2: Summary o f flavonoid synthesis. This diagram shows the basic steps in which acetyl-CoA is joined with phenylalanine to form a chalcone, the simplest flavonoid.  12  The most active structure they found was 5,7,4'-trihydroxy-8-methoxyflavone, see Figure 3 (Nagai et al. 1990).  Figure 3: 5,7,4'-trihydroxy-8-methoxyflavone  Collaborations between Dr. French and Dr. Towers have done much of the work studying the effects of flavonoids on plant viruses (French et al. 1991, French and Towers 1992, Malhotra et al. 1996, Onylagha et al. 1997). In these studies, several flavonoids have been identified as exhibiting strong antiviral properties against plant viruses. One of the most notable flavonoids is quercetin (see Figure 4), which even at very low concentrations is a very strong inhibitor of both Tomato Ringspot Virus and P V X (Malhotra et al. 1996, French and Towers 1992).  OH  0  Figure 4: Quercetin  13  1.7 Biflavonoids With all the attention that monomeric flavonoids have received for their antiviral properties, it is surprising that little work has been done to investigate the antiviral effectiveness of biflavonoids. Preliminary results indicated that biflavonoids such as robustaflavone effectively inhibit P V X infection in the local lesion assay (French, 2000). Biflavonoids are dimers made up of two flavonoid units. The best known sources of biflavonoids are the needles, bark and seeds o f gymnosperms, the leaves of Ginkgo biloba and the nuts and seeds of plants in the family Guttiferae. Biflavonoids are a very consistent character of the Psilotales, Selaginellales, Cycadales and Gymnosperms (with the exception of Gnetales and Pinaceae) (Geiger et al. 1997). In these plant groups, almost all synthesized flavonoids are shunted into biflavonoid production and the types of biflavonoids made in these plants are very consistent (Geiger et al. 1997). In the Angiosperms, the occurrence of biflavonoids is erratic and thought to have evolved several times independently. The persistence of biflavonoids in the ancient taxa mentioned above and their continued reoccurrence in the more modern taxa suggests that these compounds contribute to the fitness of these plants (Geiger et al. 1997). Because biflavonoids have been shown to be most abundant in the cuticle and outer surfaces of plants, it is thought they might provide protection against the invasion of microorganisms or herbivorous insects (Gadek et al. 1984). The first report of a biflavonoid containing antiviral activity occurred in 1992 when a screen for antiviral activity in Cephalotaxus drupacea indicated that ginkgetin, a biflavonoid originally isolated from Ginkgo biloba, was active against Herpes Simplex Virus-1 (Hayashi et al. 1992). In 1997, L i n et al. published a report indicating antiviral  14  activity of biflavonoids and characterized this group of chemicals as having antiviral properties against animal viruses. During an examination of the anti-HIV properties of Garcinia multiflora, Lin et al. noticed a high level of biflavonoids in the active fraction. Upon investigation, eight of the eleven biflavonoids screened were found to be inhibitors of HIV-1 (Lin, Anderson et al. 1997). One of these biflavonoids tested, robustaflavone, showed particularly promising results, since it had a much lower IC50 for H I V than for human cells, indicating a selective targeting of the virus. Further investigation showed robustaflavone also to be a potent inhibitor of hepatitis B (Lin, Zembower et al. 1997). These results suggested that biflavonoids might effectively inhibit a wide spectrum of viruses. Subsequently, L i n et al. again screened these same biflavonoids, this time against 11 different pathogenic viruses, testing for a broader range of antiviral activity (Lin et al. 1999). O f the ten biflavonoids they isolated from Rhus succedanea and Garcinia multiflora, all showed antiviral activity against at least one of the human viruses tested. From this study, robustaflavone, amentoflavone, hinokiflavone and agathisflavone showed high activity against several human viruses including influenza A & B , H S V 1 & 2 and measles (Lin et al. 1999). Because of this wide spectrum activity found in so many biflavonoids, it seems logical that other biflavonoids are also active; some biflavonoids may even be more selective (nontoxic to host cells) and more effective than these examined compounds. It also seems likely that these biflavonoids are effective at inhibiting more viruses than the few that were tested. The first biflavonoid to be discovered was ginkgetin, which was isolated from Gingko biloba in 1929 and identified in 1941. A s mentioned earlier, this was also the first biflavonoid to be tested and shown to be antiviral. Currently, only approximately  15  100 biflavonoids have been described; of those tested, most have shown antiviral properties.  1.8 Mechanism o f Action of Biflavonoids Very little work has been done on the mechanism of inhibition caused by antiviral biflavonoids.  During an investigation into the ability of robustaflavone to inhibit  hepatitis B , L i n et al. monitored viral D N A , m R N A and protein antigen levels and found robustaflavone to significantly lower intracellular and extracellular viral D N A levels (Lin, Zembower et al, 1997). This decrease in viral D N A suggests that robustaflavone inhibits H B V D N A polymerase. This form of inhibition is most likely to "occur during the early stages of viral genome replication such as during the formation of the primer for negative-strand D N A synthesis" (Lin, Zembower et al, 1997). Due to the large size of biflavonoids, it is unlikely that they are capable of penetrating into the core of mature viral particles. More likely biflavonoids inhibit the virus during the replication process, which takes place in the host cytoplasm. Some biflavonoids have been shown to inhibit HIV-1 reverse transcriptase in vitro (Lin, Anderson et al. 1997). It is also possible that the biflavonoids are encapsulated inside viruses that have managed to undergo genome replication (Lin, Zembower et al, 1997).  1.9 Bryophytes Recent evidence has shown that bryophytes are a rich source of biflavonoids. Bryophytes are a division of small, nonvascular, spore bearing plants with a gametophytic life cycle. The division Bryophyta include the liverworts (Hepaticae),  16  hornworts (Anthocerotae), sphagnum (Sphagnopsida), hair-cap mosses (Polytrichopsida), the lantern mosses (Andreaeopsida), and the toothed mosses (Bryopsidd). The biflavonoid content of some mosses has been shown to be as high as 2.5% of their dry weight (Zinsmeister et al. 1996). This is somewhat surprising as bryophytes, particularly the true mosses, are thought to produce very few secondary metabolites (Asakawa, 1995), and are rarely studied for their chemical properties. Nevertheless, in 1987, Hans Geiger isolated a new biflavonoid (5'-hydroxyamentoflavone) from the moss Plagiomnium elatum and showed biflavonoids to be present in seven unrelated bryophyte taxa (Geiger, 1988). More recently, Tassilo Seeger performed a 2 D - T L C screen of 200 moss species from 61 different families and reported the presence of previously known bi- and triflavonoids in 40 of these families (Seeger, 1992). Unfortunately, Seeger has not been able to publish this work as the method of 2 D - T L C does not provide definitive identification of these compounds. A s a result of Seeger's work, Zinsmeister et al. have undertaken the task of using N M R to examine these bryophytes for the presence of biflavonoids. Currently, they have tested representative species from six unrelated families of mosses and in each of these families they have found a high occurrence of biflavonoids (Zinsmeister et al. 1996). The occurrence of biflavonoids in these unrelated mosses provides evidence that biflavonoids are widespread amongst bryophyte species. Unlike the biflavonoids found in the non-flowering land plants which are very consistent, the bryophytes contain a very diverse array of biflavonoids, many of which are unique to one species (Geiger et al. 1997). Several of these unique bryophyte biflavonoids are listed in Table 1 with the mosses in which they are found.  17  Table 1: Some unique bryophyte biflavonoids. This table lists some of the biflavonoids that are reported to be unique to bryophytes.  Biflavonoid  Bryophyte found in  Literature source  5 '-Hydroxy amentoflavone 5 '-Hy droxyrobustaflavone 2,3-Dihydro-3',3"'-Biapigenin 3"'-Desoxydicranolomin 2,3-Dihydro-3"'Desoxydicranolomin 2,3-Dihydro-5',3"'Dihydroxyrobustaflavone Bartramiaflavone Campylopusaurone 5',3'"-Dihydroxyrobustaflavone 5',3'"-Dihydroxyamentoflavone  Plagiomnium elatum Rhytidiadelphus squarrosus Homalothecium lutescens Plagiomnium undulatum  Geiger, 1988  Bartramia pomiformis Campylopus clavatus Campylopus Holomitrium  Seegere/a/. 1991 Geiger & Markham,  5',3'"-Dihydroxyrobustaflavone  Hylocomium splendens  Becker et al. 1986  Seegerefa/. 1990 Seegerefa/. 1993 Rampendahl et al. 1996  The bryophyte biflavonoids have yet to be examined for any type of biological activity including antiviral activity. Bryophytes have often been overlooked when it comes to phytochemical studies, as their study poses several unique challenges.  Bryophytes do  not yield large amounts of chemicals per weight; thus in comparison to vascular plants, larger amounts of plant material is needed. Obtaining adequate amounts of material can be a challenge as bryophytes are small organisms that often grow amongst other species. Also, bryophyte identification can be problematic. Due to limited knowledge of bryophytes, these organisms are often overlooked. Currently, only one study is known to have investigated the antiviral activity of bryophytes (Kothyari, 1997). This is surprising when we consider the amount of research that has been devoted to the antiviral properties of higher plants. Unlike higher plants, mosses lack advanced structures such as thick outer layers and waxy cuticles, which are capable of providing mechanical defenses against viruses. Despite this apparent  18  vulnerability, there are no reported cases of viruses capable of infecting bryophytes and thus it seems quite possible that bryophytes contain a chemical defense against viruses. In 1997, Kothyari screened extracts from Himalayan bryophytes for activity against Tobacco Mosaic Virus and Tobacco Ring Spot Virus. O f the 31 species screened, five showed antiviral activity with the extract from Pellia epipylla showing the greatest activity (Kothyari, 1997). These findings provide even further motivation for the investigation of bryophyte biflavonoids for antiviral activity. It is estimated that there are 14,000 bryophyte species belonging to 750 genera growing in British Columbia. O f these 14,000 species, only 320 have been mentioned in chemical studies and there are 580 genera that have yet to be chemically investigated (Asakawa, 1995). With such an abundant and diverse biflavonoid composition produced in these plants which produce very few other secondary metabolites (Geiger et al. 1997, Asakawa 1995), bryophytes are an excellent candidate for the study of biflavonoids.  1.10 Objectives The goal of this project was to identify new sources of antiviral compounds. N e w antiviral compounds are of great interest to those involved in producing virus-free crops. It is hoped that compounds can be found that are less toxic and less expensive than ribavirin, thus being of greater agricultural use. The ultimate goal is to find an antiviral substance that can be used to exterminate viruses from existing crops in the field. In this project, P V X is used as a model for plant viruses; however, it is very likely that substances identified by this project will be useful against many other viruses, just as ribavirin is quite useful in the treatment of several plant and human viral diseases.  19  There is mounting evidence that indicates biflavonoids are very strong viral inhibitors and effective against a great many viruses. It was the goal o f this project to further study the antiviral properties of biflavonoids as they relate to P V X . With the goal of identifying new sources of antiviral activity and the high occurrence of biflavonoids present in bryophytes, this project investigated the antiviral properties of extracts from this very interesting, but often understudied group of plants.  20  2 Methods 2.1 Collection and Identification of Bryophytes The bryophyte material used for the antiviral screening was collected from various locations throughout British Columbia between August 2000 and September 2001. Collected specimens were rinsed clean, dried thoroughly and stored in paper bags at room temperature. Approximately one to ten grams of dried material were collected for each sample. Identification was performed using Crum's text "The Mosses of Eastern North America" (Crum, 1981) and Schofield's handbook "Some Common Mosses of British Columbia" (Schofield, 1992). Identified specimens were then verified through comparison with material preserved in the U B C Herbarium. Voucher specimens are stored in the U B C Herbarium, Vancouver, B C . Table 2: Bryophyte collection. The following 50 bryophyte samples were used in the a n t i - P V X assay.  Collec. No.  Name  Date  Location  Collection notes  107  Andreaea nivalis Hook.  Sep 1, 2001  Squamish, B C . Garibaldi Park.  Growing on rocks on north facing alpine meadow.  128  Atrichum selwynii Aust.  May 15, 2002  North Vancouver, B C Mosquito Creek.  Growing near forest trail  129  Atrichum undulatum (Hedw.) P. Beauv.  May 15, 2002  North Vancouver, B C Mosquito Creek.  growing along trail  67  Bryum argenteum Hedw.  Aug 24, 2000  Prince George, B C  growing on roof top  144  Bryum capillare Hedw.  June 22, 2002  Near Port Alberni, B C Kennedy Lake  Growing on rocks  80  Bryum miniatum Lesq.  May 7, 2001  Nanaimo, B C . End of cranberry road.  A red moss growing on a very wet rock field in direct sunlight.  85  Buckiella undulatum Hedw.  July 31, 2001  Vancouver, B C . Pacific Spirit Park.  Growing amongst large amount of pine • needles. Individuals small and flattened.  94  Buckiella undulatum Hedw.  Aug 6, 2001  Port Alberni, B C . Sproat Lake.  Growing on forest floor. Unusually large fluffy appearance.  21  134  Claopodium crispifolium (Hook) R. & C .  May 16, 2002  North Vancouver, B C Mosquito Creek.  Growing on side of Maple tree  103  Dfcranum Turner.  Sep 1, 2001  Squamish, B C . Garibaldi Park.  Found on Ground and stumps  83  Eurhynchium (Sull.) Jaeg.  July 31, 2001  Vancouver, B C Pacific Spirit Park, 16th Ave.  A feather moss growing on a large rock.  150  Eurhynchium praelongum (Hedw.) Br.Eur.  July 31, 2001  Vancouver, B C Pacific Spirit Park.  Growing on log.  160  Eurhynchium praelongum (Hedw.) Br.Eur.  Sep 1, 2001  Squamish, B C Garibaldi Park.  O n boulders by stream.  68  Grimmia pulvinata (Hedw.) Sm.  Aug 28, 2000  Stein Valley, B C  Growing on alpine boulders.  88  Hylocomium splendens (Hedw.) B . S . G .  Aug 6, 2001  Port Alberni, B C Sproat Lake.  Plants growing to very large size, dominant ground cover.  127  Hylocomium splendens (Hedw.) B . S . G .  May 15, 2002  North Vancouver, B C Mosquito Creek.  Dominant ground cover. Collected for chemical study  84  Hypnum circinale Hook.  July 31, 2001  Vancouver, B C Pacific Spirit Park, 16th Ave.  A small moss growing flattened along decaying log.  105  Hypnum circinale Hook.  S e p 1, 2001  Squamish, B C Garibaldi Park.  Shaded area, on log near trail.  109  Hypnum circinale Hook.  Sep 5, 2001  Vancouver, B C Lighthouse Park.  Growing on dry rock, exposed area.  91  Isothecium stoloniferum Brid.  Aug 6, 2001  Port Alberni, B C Sproat Lake.  Horizontal form. Found growing on top of a log. Feathery growth form.  92  Isothecium stoloniferum Brid.  Aug 6, 2001  Port Alberni, B C Sproat Lake.  Vertical form. Found hanging in hair like form off of deciduas trees.  89  Leucolepsis acanthoneuron (Schwaegr.) Lindb.  Aug 6, 2001  Port Alberni, B C Sproat Lake.  Growing to very large size.  136  Leucolepsis acanthoneuron (Schwaegr.) Lindb.  May 16, 2002  North Vancouver, B C Mosquito Creek.  Growing on soil in shaded area.  124  Oligotrichum aligerum Mitt.  May 15, 2002  North Vancouver, B C Mosquito Creek.  Found on ground near trail.  137  Plagiomnium TJ Kop.  May 16, 2002  North Vancouver, B C Mosquito Creek.  Growing on shaded forest floor.  110  Pleurozium schreberi (Brid.) Mitt.  S e p 1, 2001  Squamish, B C . Garibaldi Park.  Growing on dead wood, near lake.  106  Pogonatum contortum Menzies ex Brid.  Sep 1, 2001  Squamish, B C . Garibaldi Park.  Growing on forest floor.  146  Pogonatum contortum Menzies ex Brid.  June 22, 2002  Sutton Pass Vancouver Island.  Growing on stream bank and in water.  69  Polytrichastrum alpinum (Hedw.) G L S m .  Aug 28, 2000  Stein Valley, B C  Found in clearing next to logging road on sand and pebbles  139  Polytrichum Hedw.  May 16, 2002  North Vancouver, B C Mosquito Creek.  Growing on forest floor.  fuscescens  oreganum  insigne (Mitt.)  commune  22  June 22, 2002  Near Port Alberni Kennedy Lake  Growing beside logging road, in exposed area  Polytrichum piliferum Hedw.  May 15, 2002  North Vancouver, B C Mosquito Creek.  Growing on forest floor  Racomitrium canescens (Hedw.) Brid.  S e p 1, 2001  Squamish, B C . Garibaldi Park.  Found on ground beside trail  71  Racomitrium canescens var. ericoides (Brid.) B . S . G .  Aug 29, 2000  Pemberton, B C . Duffy Lake Rd. Lillooet Lake.  Growing on rock in very dry dusty area. Distinctly grey when dry. Bushy with many leaves, very full appearance.  121  Racomitrium lanuginosum (Hedw.) Brid.  April 15, 2002  Vancouver, B C Cypress Park  Growing on rocks.  131  Rhizomnium glabrescens (Kindb.) T J Kop.  May 16, 2002  North Vancouver, B C Mosquito Creek.  Growing on Rotten logs  126  Rhytidiadelphus loreus (Hedw.) Warnst.  May 15, 2002  North Vancouver, B C Mosquito Creek.  Growing on old log.  120  Rhytidiadelphus squarrosus (Hedw.) Warnst.  April 13, 2002  Vancouver, B C UBC  Growing amongst grass.  95  Rhytidiadelphus triquetrus (Hedw.) Warnst.  Aug 6, 2001  Port Alberni, B C Sproat Lake.  Forming dense mats on forest floor. Shaded area.  70  Rhytidiadelphus triquetrus (Hedw.) Warnst.  Aug 28, 2000  Stein Valley, B C  Growing on rocks  101  Rhytidiopsis robusta (Hook.) Broth.  Sep 1, 2001  Squamish, B C . Garibaldi Park.  Forming dense mats on Forest floor. Dominant species in Subalpine forest.  93  Scapania bolanderi Austin.  Aug 6, 2001  Port Alberni, B C Sproat Lake.  Growing on vertical surface of a stump.  57  Sphagnum capillifolium (Ehrh.) Hedw.  Aug 21, 2000  Near Prince George, B C Ness Lake  Forming a floating mat in a marsh,  61  Sphagnum fuscum (Schimp.) Klinggr.  Aug 21, 2000  Near Prince George, B C Ness Lake  Growing in spruce bog.  58  Sphagnum Flatberg.  pacificum  Aug 21, 2000  Near Prince George, B C Ness Lake  Margin of pool in pine forest.  63  Sphagnum Lindb.  papillosum  Aug 21, 2000  Near Prince George, B C Ness Lake  Growing in a wet bog area among other species  62  Sphagnum rubellum Wils.  Aug 21, 2000  Near Prince George, B C Ness Lake  Growing in a wet bog area among other species  64  Sphagnum Warnst..  Aug 21, 2000  Near Prince George, B C Ness Lake  Growing on refuse in Bog  132  Tetraphis pellucida Hedw.  May 16, 2002  North Vancouver, B C Mosquito Creek.  Growing on rotting stump  119  Tortula muralis Hedw.  April 13, 2002  Vancouver, B C UBC  growing on exposed concrete  145  Polytrichum Hedw.  123 100  juniperinum  russowii  23  2.2 Extraction of Bryophytes Clean air-dried plant samples were ground in liquid nitrogen using a mortar and pestle. The resulting fine ground powder underwent three consecutive two day extractions at room temperature using approximately 40mL of ethanol per extraction. Upon each extraction the supernatant was vacuum filtered and dried in vacuo. The resulting crude extracts were stored at -20°C.  2.3 The Antiviral Assay Extracts and compounds were tested for antiviral activity using a local lesion assay with P V X infection of Chenopodium quinoa, as described previously by French et al. 1991. In this assay the bryophyte extract or other test substance is incubated with P V X for 30 minutes before being used to inoculate one side of a C. quinoa leaf. The remaining half leaf is then inoculated with a control solution containing an equivalent P V X concentration. After a week of incubation in a growth chamber (20°C, cool white light, 16h light: 8h dark) the plant develops lesions in response to P V X infections. The percent inhibition caused by the test substance can then be calculated by comparing the number of lesions on each side of the leaf. Test extracts were prepared as solutions containing lOmg/mL of dried extract in 5% Ethylene Glycol Monomethyl Ether ( E G M E ) . E G M E was used to improve the solubility of the extract in the solution; at these concentrations this solvent has been shown to have no effect on the number of lesions produced by P V X (French and Towers, 1992). To avoid precipitation in water, the dried extract was first dissolved in E G M E and then distilled water was added.  24  To incubate the virus with the extract solution, 20uL of a 1.15mg/mL P V X solution was added to 200uL of the lOmg/mL extract solution, resulting in a final viral concentration of O.lOmg/mL and a final extract concentration of 9.1 mg/mL. A control solution was prepared in a similar manner, except the extract solution was replaced by 200uL of 5% E M G E . Both the control and test solutions were incubated for 30 minutes at room temperature. Following incubation of the test and control solutions, the solutions were used to inoculate five week old C. quinoa. Plants which were older than five weeks were no longer suitable for use in the assay as they possessed a higher level of resistance to viral infection and thus did not produce enough lesions to yield consistent results. Prior to inoculation, plants were powdered with carborundum in order to abrade the leaf during the inoculation process, thus allowing transmission to occur. The inoculation was performed using a freshly gloved finger. The control side of the leaf was chosen at random and then marked by punching a small hole on that side of the leaf tip. On each side of the leaf, 1 OuL of the corresponding solution was rubbed into the leaf. Typically each plant possessed 4-7 leaves suitable for inoculation.  25  Figure 5: Five week old Chenopodium quinoa. Following one week of incubation in an environmental chamber the resulting lesions on each half leaf were counted. Leaves that did not produce a minimum of 15 lesions on the control side were not counted, as often their results were not consistent. The percent inhibition for each leaf was calculated and the average percent inhibition and standard deviation on all leaves was also calculated.  26  Figure 6: Chenopodium quinoa leaf showing lesions. PVX induced lesions are present on control side of leaf after one week of incubation.  Each sample was tested on at least three separate occasions, each time using a minimum of four leaves on each of three plants. Leaves failing to produce 15 lesions on the control half were not counted. If multiple leaves of a plant failed to produce this minimum level of infection, then the results of the plant were ignored, as it is likely the plant was overly mature and possessed an increased resistance to infection. The results of samples which did not yield a minimum of three replications involving three plants each, have been deemed unreliable and are not reported.  27  C. quinoa plants were grown from seed in an environmental growth chamber with 16 hour photosynthetic periods at 25°C. Approximately 50 seeds were germinated in a three inch plastic pot filled with Terralite® Readi-Earth® potting soil. One week after seeds were sown, seedlings were transplanted into individual three inch plastic pots.  2.4 Biflavonoid Testing Four purified biflavonoid compounds: robustaflavone, hinokiflavone, amentoflavone and 7,4',4'-amentoflavone were obtained and tested for antiviral activity using the local lesion assay. The robustaflavone sample was provided by Yuh-Meei L i n and the MediChem research group. The remaining three biflavonoids were purchased from Apin Chemicals Ltd., U . K . Due to the small amount of material available, samples were only successfully tested at a final concentration of 1.38mg/mL.  2.5 Fractionation o f the Bryophyte Hylocomium splendens A large quantity of H. splendens was collected from North Vancouver highlands. The sample was dried and ground to a fine powder using a mechanical grinder. The resulting 600 gram sample was subjected to four consecutive three day extractions in boiling ethanol. The resulting dark green coloured extract was then dried down in vacuo at 40°C to yield 56.1 grams of powdered crude extract. A liquid-liquid partition was performed in order to separate the crude extract into four fractions based on solubility of the different components within the crude extract. This partition was performed by dissolving the crude extract in water, placing the solution in a two liter separatory funnel and washing several times with 95% hexane,  28  followed by several washings with ethyl acetate and finally the remaining solution was washed with butanol. Thin Layer Chromatography (TLC) was used to analyze the composition of each fraction. Due to similar T L C profiles, the hexane and the ethyl acetate fractions were amalgamated. The resulting three fractions were then tested for anti-PVX activity using the local lesion assay as was described previously. Following the results of the local lesion assay, further separation of the hexaneethyl acetate fraction was achieved through the use of a dry loaded silica gel column. 24.16g of the hexane-ethyl acetate fraction were dissolved in acetone and dried onto 67g of SiliCycle® silica gel 60 (230-400 mesh). The sample was then loaded on to a 3.81cm diameter column which was packed with 400g of the same silica gel. The column was then washed with solvent beginning with 100% hexane and the polarity of the solvent was gradually increased by diluting the hexane with acetone in 5-10% increments. Upon addition of 100% acetone, the column was terminated and allowed to run dry. The resulting 115 fractions were then analyzed using thin layer chromatography and similar fractions were amalgamated to yield 30 unique groups of fractions. Based on the polarity, colour and quantity, seven of the amalgamated groups were then chosen for antiviral testing. The three adjacent groups that were identified as being active were amalgamated and with the assistance of Dr. Dong Sheng Ming, separated in a Sephadex™ LH-20 column eluted with methanol.  29  3 Results & Discussion 3.1 Anti-viral Activity of Bryophytes. Table 3 provides an alphabetical listing of the percent inhibition of P V X caused by each bryophyte sample that yielded consistent results. The percent inhibition has been calculated as an average of all testing for each sample. The percent error has been calculated as the standard deviation among all leaves tested for each sample. 3.1.1  Results were not obtained from all samples collected. O f the 70 samples that were collected and identified, reportable data was only  obtained from 50 of the samples. Leaves which did not produce a minimum of 15 lesions on the control side were not counted, as the results were too easily distorted by a small number o f inconsistent lesions; as well, the standard deviation amongst these leaves was very high. Data was also not taken from plants that contained multiple leaves that failed to develop adequate lesions. It was noted that the lesion counts from the remaining leaves on these plants did not correspond to data taken from other plants. The failure o f these plants to produce adequate lesions is most likely due to the plants being at a higher maturity level and thus having a greater resistance to infection. Ideal growing conditions and experience are required in order to grow large numbers of C. quinoa all possessing the exact same fitness and maturity. In many cases, experiments failed to produce adequate numbers of lesions and had to be repeated. These additional repetitions were not anticipated at the time o f collection and thus several samples were depleted before useful data could be obtained.  30  Table 3: The anti-PVX activity of tested bryophyte species. All samples were tested at 9.lmg/mL using the local lesion assay. The bars on the right are a graphical representation of the levels of inhibition and error for each sample. Collection # Bryophyte Species Andreaea nivalis 107 Atrichum selwynii 128 Atrichum undulatum 129 Bryum argenteum 67 Bryum capillare 144 Bryum miniatum 80 Buckiella undulatum 85 Buckiella undulatum 94 Claopodium crispifolium 134 Dicranum fuscescens 103 Eurhynchium oreganum 83 Eurhynchium praelongum 150 Eurhynchium praelongum 160 Grimmia pulvinata 68 Hylocomium splendens 88 Hylocomium splendens 127 Hypnum circinale 84 Hypnum circinale 105 Hypnum circinale 109 Isothecium stoloniferum 91 Isothecium stoloniferum 92 Leucolepsis acanthoneuron 136 Leucolepsis acanthoneuron 89 Oligotrichum aligerum 124 Plagiomnium insigne 137 Pleurozium schreberi 110 Pogonatum contortum 146 Pogonatum contortum 106 Polytrichum commune 139 Polytrichum piliferum 123 Polytrichastrum alpinum 69 Polytrichum juniperinum 145 Racomitrium canescens 71 Racomitrium canescens 100 Racomitrium lanuginosum 121 Rhizomnium glabrescens 131 Rhytidiadelphus loreus 126 Rhytidiadelphus squarrosus 120 Rhytidiadelphus triquetrus 95 Rhytidiadelphus triquetrus 70 Rhytidiopsis robusta 101 Scapania bolanderi 93 Sphagnum papillosum 63 Sphagnum russowii 64 Sphagnum capillifolium 57 Sphagnum fuscum 61 Sphagnum pacificum 58 Sphagnum rubellum 62 Tetraphis pellucida 132 Tortula muralis 119  % inhibition  % error  98 98 100  6  26 38 100 100 91 65 62 72 99 99 100 86 89 71 44 96 43 71 91 92 94 100 90 98 64 91 61 99 100 87 90 98 98 99 94 97 100 93 93 77 100 61  17 36 59 14 10 40 26 29 38  23 25 61 19 64 32 19 12 14 16 41 18  10  21 12 -10  45  100  31  3.1.2  The majority of species caused a high level of inhibition. O f the 41 species successfully tested for antiviral activity, 29 resulted in greater  than 75% inhibition against P V X . Thus, 71 % of the bryophyte species examined were highly effective at inhibiting P V X infections in C. quinoa when tested at 9.1mg/mL. In comparison to another antiviral screening of this nature, this study has identified a very high proportion of its samples as being antiviral. In a very similar screening performed in this lab, 31 algal species were tested for P V X inhibition at a concentration of lOmg/mL in a similar local lesion assay. O f the species tested only seven species (23% of those tested) were found to cause greater than 75% P V X inhibition (Pardee, 2001). 3.1.3  The correlation between activity and error. Despite the fact that 46 of the 50 samples listed in Table 3 caused greater than 25%  inhibition against P V X , only 29 have been labeled as highly active. The labeling of samples as "highly active" was restricted to samples exhibiting greater than 75% inhibition, as samples causing less than this level of inhibition exhibited a greater level of experimental error. B y rearranging the samples in terms of their reported antiviral activity, as is done in Figure 7, it becomes apparent that there is a definite inverse correlation between the degree of inhibition and the level of uncertainty in a sample. This relationship between error and inhibition is a common characteristic of the local lesion assay (Pardee, 2001). A s the level of inhibition decreases, the outcome becomes more, random and the deviation among the results increases.  32  Coll. No  Bryophyte Species  -40.0  Percent inhibition of PVX  Figure 7: Relationship between inhibition and uncertainty i n bryophyte samples. Data from Table 3 is arranged in order of antiviral activity. As activity decreases uncertainty increases.  33  3.1.4  High error is a result of inconsistent infection production. The high level of error present in the less active samples indicates the inconsistency  in the number of infections which occur upon each inoculation. Table 4 illustrates this point by displaying the local lesion assay results of sample 127 Hylocomium splendens (Hedw.) B.S.G. The total inhibition for this sample was calculated as 99.5% with a standard deviation of 1.8%. It would appear that the results of the experiments for this sample are very consistent; however, i f we compare the number of lesions on each control for this sample, we see that they have a standard deviation of 21.9%. Thus the inoculation of the same control solution has a fairly inconsistent outcome. This same inconsistency is noted among all other data obtained from the local lesion assay. Therefore, in the case of a non-active sample, we obtain a high level of uncertainty, as the number of lesions produced by each inoculation is not consistent. Despite this inconsistency in the number of lesions caused by an inoculation, highly antiviral substances still produce very consistent results as they prevent the formation of lesions at any level.  34  Table 4: The local lesion assay results of sample 127 H. splendens. The number o f lesions counted on the control and test side o f each leaf are listed. The percent inhibition o f each leaf has been calculated. The results o f all leaves have been averaged and listed as the average percent inhibition. The standard deviation o f all controls has been calculated to show the variable levels o f lesion development. Leaves producing less than 15 lesions were omitted.  Experiment 3  Experiment 2  Experiment 1 Plant 1 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 20 20 75 15 70 20  test 0 0 5 0 5 0  % inhibition  Plant 2 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 35 60 15 20 18 15  test 0 0 0 0 0 0  % inhibition  Plant 3 leaf! leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 16 15 20 16  test 0 0 0 0  % inhibition  100.0 100.0 93.3 100.0 92.9 100.0  100.0 100.0 100.0 100.0 100.0 100.0  100.0 100.0 100.0 100.0  Plant 1 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 25 77 55 20 72  test 0 3 0 0 0  % inhibition 100.0 96.1 100.0 100.0 100.0  Plant 1 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 50 68 65 15 38 24  test 0 0 0 0 0 0  % inhibition 100 100.0 100.0 100.0 100.0 100.0  Plant 2 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 45 43 17 80 16  test 0 0 0 0 0  % inhibition 100.0 100.0 100.0 100.0 100.0  Plant 2 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 43 67 15 25  test 0 5 0 0  % inhibition 100.0 92.5 100.0 100.0  Plant 3 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 70 20 44 20 18  test 0 0 0 0 0  % inhibition 100.0 100.0 100.0 100.0 100.0  Plant 3 leaf 1 leaf 2 leaf 3 leaf 4 leaf 5 leaf 6  control 17 34 25 60 22  test 0 0 0 0 0  % inhibition 100.0 100.0 100.0 100.0 100.0  A\ /erage percent inhibition Standard deviation of inhibition  99.5 % 1.8 %  Standard deviation of control  21.9 %  35  3.1.5  Eight species with multiple samples. Only 41 different bryophyte species were represented by the 50 samples which  yielded results. Additional samples of a particular species were chosen due to observable differences within samples of the same species. These samples may have differed in growth form, habitat, or samples were different varieties of a species. Table 2 shows specific details or differences for each sample. Despite the observed phenotypical differences between the repeated samples, they produced very similar results. The two samples of Racomitrium canescens, were an exception to this as they yielded very different results one producing 63.6% inhibition and the other producing 90.6%. Unlike other repeated samples, the Racomitrium canescens samples were identified as different varieties of the species. The less active sample was identified as Racomitrium canescens var. ericoides (Brid.). It is possible that the differences separating these two varieties are greater than they appear and go beyond physical appearances. The other repeated samples that produced similar results provide evidence to support the reproducibility of the methods used in this study. The fact that different samples of the same species which were collected during different seasons and from very different habitats still produced the same effect, increases the likelihood that the reported results are indicative of the true antiviral properties of these species. 3.1.6  Relatedness among bryophyte samples. Figure 8 shows the phylogeny of the bryophyte samples with their reported antiviral  activity. The phylogenetic tree lists the 41 species and the corresponding 26 genera and 14 families to which they belong. This sample is small when compared to the estimated 400 families, 750 genera and 14000 species of bryophytes found in British Columbia.  36  When the relationships between bryophyte species are taken into consideration, it becomes apparent that the activity of the samples in Table 3 is concentrated among groups of related species. The majority of the active samples belong to the three families: Hylocomiaceae, Polytrichaceae and Sphagnaceae. A l l of the samples from these families caused high levels of viral inhibition. Because a minimum of six different species exhibited very high levels of viral inhibition, it is possible that anti-viral properties are common throughout these families. Other bryophyte families tested may also have widespread anti-viral activity, but due to poor representation of their families in this study, such predictions cannot be made at this time. The phylogenetic tree also indicates several families that contain poor antiviral activity. The Mniaceae, Bryaceae and Brachytheciaceae all contain several species with little antiviral activity and do not seem to be noteworthy in terms of their antiviral properties. Bryophytes were initially chosen for this study due to their abundance of biflavonoids; however, not all bryophytes produce these compounds. In fact, it seems that the presence of biflavonoids is restricted to the Bryopsida or toothed mosses. The Sphagnopsida and the Andreaeopsida are quite unique in that they do not produce any flavonoids (Geiger et al. 1997). While the Polytrichopsida have not been as intensely studied, as of yet, no flavonoids have been found in this group either (Geiger et al. 1997). In terms of chemistry, the Hepaticopsida are the most thoroughly studied of the bryophytes. While this group is known for its diverse secondary metabolites; no biflavonoids have been found to be present (Asakawa 1995). In view of the theory that the bryophytes would contain antiviral activity because of their abundance of biflavonoids, we would not expect the Andreaeopsida, Sphagnopsida, Hepaticopsida and  37  possibly the Polytrichopsida to be active. Despite their lack of biflavonoids, all representatives of these groups caused extremely high levels of P V X inhibition. It is possible that this antiviral activity in these latter groups is due to other antiviral compounds such as tannins, which are present in these groups, but absent from the Bryopsida. The Sphagnopsida are well known for their tannin content and it is not surprising that they contain these large antiviral compounds, as there is great selective pressure from their aquatic habitat for antiviral compounds such as this. Despite their reported abundance of biflavonoids, several groups of the Bryopsida did not exhibit antiviral activity. Many of the non active species have been explicitly cited to contain biflavonoids and thus it is possible that not all of these compounds are active against P V X . However it is also possible that some of the biflavonoids present in these plants could not be extracted by the methods used. Several mosses have been shown to contain a high content of biflavonoids which are bound within the cell wall; these compounds cannot be extracted with alcohol (Geiger et al. 1997). It is also possible that the bryophytes were harvested during a time in which they possessed extremely low levels of biflavonoids. Studies have shown that the flavonoid content of mosses undergo considerable seasonal variation (Brinkmeier, 1999). In view of this, it is possible that the non-active species did not possess considerable quantities of biflavonoids at the time these samples were harvested.  38  O  snntNr-ontNOO  T T T. <0  JS  iI I§i *•li ff li 1 E  S S  1 3 f j a -I § s  r  [ 1i  E N  a  £  E £  E  Ti  E  «  |  CO CO  c  Q. >. I  cu  O  o.S2  •CCD  eoc  GCL< • I i  CO  o .2 E E •c O  ® ® CO  •HOC OCL< 0) CD  CD  E  •o  t  O  0.  CD CO  •g c  CD O  Q  5  CD CD  <  £ CA 0) CD JZ  o  •c  %  co  c  <  CO  ca •o a  CD  CD CO  •8 o a. CO  o .c o •c  1o a.  8 CO cU> CO  .C  a.  CO  o c_ 3  CD CD  CO  CD CD  I 2  8 CO -g j= a  o  0.  J CO  O <D O. I -  CD CO  •g  JC  a  CD .CD  I  T 3  f l 11  _1_  x: o  CO  CO  2  — c o o  sCO  s  'E E  .2  E  •2 8"  O£ < < L_L  a>  O  H i t III si I I I  8  E  lit 1 CD  3  IV Ii I' T i  S3 S  CO  9 -* =  C •  tO K CM CO  CO  c  CO Ql CO  0  ca •g 'c O) CO  a.  CO  CO  T3  a o c  O) CO  .c a  CO  39  3.2 Anti-viral Activity of Biflavonoids.  Four isolated biflavonoids compounds were obtained and tested for antiviral activity using the local lesion assay and C. quinoa. These compounds are known to be present in the vascular plants Rhus succedanea and Garcinia multiflora and have previously been tested against human pathogenic viruses (Lin, Anderson et al. 1997). The compounds were only available in very small quantities and thus were first tested at the low concentration of 1.38mg/mL; a concentration at which other pure compounds are commonly tested using the local lesion assay (French and Towers, 1992). Once data was successfully obtained at this low concentration, an attempt was made to concentrate the remaining biflavonoids solutions and test them at 13.8mg/mL. Unfortunately this data could not be reported in this study as several trials did not produced adequate lesions and the limited material did not allow for further testing. However, preliminary results were very unusual; at higher concentrations these compounds appeared to increase the level of P V X infection. Table 5 reports the results of testing the biflavonoid compounds at 1.38mg/mL. Robustaflavone caused the greatest level of viral inhibition of the biflavonoids tested. Hinokiflavone was also shown to exert a very high level of inhibition. Amentoflavone7,4',4'trimethyl ether produced minimal levels of inhibition and amentoflavone was not active.  40  Table 5: The anti-PVX activity of the biflavonoid compounds. The following four biflavonoids were obtained as pure isolated compounds and tested at 1.38mg/mL using the local lesion assay. Robustaflavone and Hinokiflavone showed high levels o f inhibition for this low concentration.  Biflavonoid  Percent inhibition  Robustaflavone Hinokiflavone Amentoflavone Amentoflavone-7,4',4'trimethyl ether  85.1 ± 2 2 . 5 78.3 ± 19.3 -10.5 ± 4 7 . 2 29.0 ± 46.6  The first three compounds in Table 5 have previously been tested for antiviral activity against animal viruses. In their 1997 study, L i n et al. tested 14 biflavonoids for Rhus succedanea and Garcinia multiflora for their ability to inhibit HIV-1 reverse transcriptase (RT). O f these biflavonoids, robustaflavone, hinokiflavone and amentoflavone were all found to cause a very high level of HIV-1 R T inhibition (9790%) when tested at the rather high concentration of 200ug/mL. The reported activity of these compounds against HIV-1 R T does not correspond to their activity against P V X in Table 5 as amentoflavone did not demonstrate any inhibition of P V X infection. While amentoflavone-7,4',4'trimethyl  ether was not tested in L i n , Anderson et al.'s 1997 study,  other methoxylated biflavonoids were. L i n et al. noticed that by methylating hydroxyl groups on these biflavonoids the compounds lost antiviral activity (Lin, Anderson et al. 1997). Based on these observations, we would not have expected amentoflavone7,4',4'trimethyl ether to have caused the 29.0% inhibition against P V X , which was observed in this study. Further work by L i n et al. tested 10 of the same biflavonoids for antiviral activity against 11 different viruses: influenza A/Texas/36/91, influenza A/Beijing/32/92, Influenza B/Panama/45/90, Measles, Adenovirus Type 5, Parainfluenza Type 3,  41  Respiratory Syncytial Virus, Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, Human Cytomegalovirus, and Varicella Zoster Virus (Lin et al. 1999). Again in this study robustaflavone, hinokiflavone and amentoflavone were among the biflavonoids tested. In all cases these three biflavonoids caused viral inhibition against the 11 human pathogenic viruses. The effectiveness of each biflavonoid varied for each virus. In view of this observation, we should not be surprised that all three of these biflavonoids were shown to be active against HIV-1 R T while only two were active against P V X . O f these three biflavonoids, amentoflavone was the least active against the 11 viruses. This corresponds with the results of the biflavonoids tested against P V X . It should also be noted that in their 1999 study L i n et al. demonstrated that in all 11 cases the biflavonoids exhibited greater inhibition against the virus while still possessing lower toxicity to the host cells than the available prescription drugs (ribavirin, 9-(3-hydroxy-2-phophonylmethoxypropyl)adenine, acyclovir and gancyclovir) (Lin et al. 1999). The structures of the four biflavonoids listed in Table 5 are provided in Figure 9 Figure 12 on page 44. A l l of these biflavonoids are dimers of the flavonoid apigenin. Robustaflavone and hinokiflavone are very similar compounds; their only difference is the way in which the apigenin units are joined. Robustaflavone has a 3'-6 carbon-carbon linkage and hinokiflavone has a 4'-6 ester linkage. This difference in attachment did not seem to correspond with any significant difference in the anti-PVX properties of these compounds; however these different structures did exhibit different levels of inhibition against several of the human viruses mentioned earlier (Lin et al. 1999). Both robustaflavone and amentoflavone are different dimers of apigenin; amentoflavone has a  42  5'-8 linkage. Because of this difference in attachment, these compounds will have a different three-dimensional shape which is most likely the cause for their different antiviral activities. This difference in attachment to the first subunit is minor as both the 5' and 3' position are meta positions have similar properties. However, the position of attachment to the second subunit will have an impact on the electron densities throughout the surrounding pi-orbitals of the compounds, as the 6 and the .8 position have different properties. The 5'-8 linkage in amentoflavone will cause the oxygen forming the cyclic ether bond in C-ring of the second apigenin subunit to possess a lower electron density when compared to that of robustaflavone. It has been noted that electron dense units such as hydroxyl groups are a key component of all active biflavonoids (Lin, Anderson et al. 1997) and thus it is possible that the decrease in electron density of the second cyclic ether in the amentoflavone molecule may contribute to its lower activity.  43  OH 0 Figure 9: Robustaflavone  OH  HQ  0  D  Figure 10: Hinokiflavone  HO  0  Figure 11: Amentoflavone  HO  Q  HO  0  Figure 12: Amentoflavone-7,4',4'trimethyl ether  3.3 The Chemistry of Hylocomium splendens Hylocomium splendens was selected for further chemical investigation. In addition to anti-viral activity, this species was selected because of its abundance and large plant size which made collection of large amounts of material possible. This plant was also chosen because it is a member of the Bryopsida, the largest and yet least chemically studied of the bryophyte groups. In addition, this plant has been very poorly studied in terms of its organic chemistry and thus any information gained would be of value. It is surprising that so little work has been done in terms of this plant's organic chemistry, when this plant has been so widely used as an environmental indicator and a great deal o f investigation has been done into this plant's inorganic chemistry. Becker et al. are responsible for the only published information regarding this species' organic chemistry (Becker et al. 1986). In a very brief study of the chemistry of H. splendens Becker et al. isolated only one compound from the plant. This highly abundant compound which they isolated was identified as a new biflavonoid, 5',3'"-dihydroxyrobustaflavone (Becker et al. 1986). In addition to this biflavonoid, Becker et al. observed six other flavonoid compounds to be present in the plant; however they were unsuccessful in isolating and identifying these compounds. The chemical study of//, splendens was particularly interesting as there has been very little work done in this area and the plant is known to possess large amounts of one biflavonoid. The main purpose of investigating the chemistry of this plant was to determine i f biflavonoids were responsible for the anti-PVX activity; however with such  45  a limited understanding of the chemistry of these mosses, any chemical information gained would be valuable. Upon liquid-liquid fractionation of the H. splendens extract, fractions were compared using thin layer chromatography. The chromatograms of the hexane and ethyl acetate fractions exhibited a very similar spotting pattern as is shown in Figure 13. In view of the similarities of these two fractions, they were amalgamated into one fraction.  Figure 13: Comparison of the hexane and ethyl acetate fractions of H. splendens. The silica plates were developed in a solution which was three parts hexane and two parts acetone. The plate on the left has been stained withl5% phosphomolybdic acid. The plate on the right has not been stained. Spots visible under normal light have been circled. The chromatographs of the hexane fraction and the ethyl acetate fractions both possess a similar spotting pattern.  The three unique fractions produced by the liquid-liquid partition were tested for activity using the local lesion assay. The hexane and ethyl acetate fractions were considerably more active than the butanol or water fractions and thus were selected for further separation using a silica column. The hexane-ethyl acetate fraction was separated  46  into 115 fractions and these fractions were again compared using thin layer chromatography. Using the chromatograms, the fractions were grouped according to their chemical similarities. Seven fractions were then selected for antiviral testing using the local lesion assay. The groups were selected as they represented the different groups which were likely to contain activity (i.e. extremely non-polar groups were not considered). The results of the antiviral assay for the different fractions are listed in Figure 14 and indicate that most of the activity in the plant is localized between fractions 74-95 of the column. Coincidently, these fractions were also unique in that they were the only fractions to possess a golden colour, which is a common characteristic of flavonoids. PVX inhibition caused by H.splendens  fractions  120  H. splenden Fraction  Figure 14: P V X inhibition caused by H. splendens fractions. The combined hexane and ethyl acetate fraction possessed the highest activity of the liquid-liquid partition. Fractions 74-95 showed the highest activity of the silica column fractions which were tested.  The three groups of fractions which were represented by fractions 74, 80-84 and 95 were amalgamated and with the assistance of Dr. Dong Sheng Ming, were further separated using a column of Sephadex™ LH-20. The resulting separation yielded only  47  one pure compound which was of a quantity large enough to be analyzed. This compound has a distinct golden colour which is characteristic of flavonoids. This sample has subsequently been sent for N M R analysis. Currently, the N M R results have not been received and the identity of this compound is not known. Unfortunately, time and materials did not allow for further investigation into the chemistry of H. splendens. Due to the small amount of compound isolated from the active fractions, this compound could not be tested further for antiviral activity. Based on the abundance of 5',3'"-dihydroxyrobustaflavone reported to be present in H. splendens (Becker et al. 1986) and based on the colour of the isolated compound, it is quite possible that the isolated compound is indeed 5',3'"-dihydroxyrobustaflavone.  HQ  Q  Figure 15: 5',3'"-Dihydroxyrobustaflavone  The structure of 5',3'"-dihydroxyrobustaflavone is displayed in Figure 15. In comparison to the other biflavonoids examined, this compound has two additional hydroxyl units on the 3' positions of each monomeric unit. The free hydroxyl units are thought to be involved in viral particle interaction (Lin, Anderson et al. 1997), thus the  48  added hydroxyl units may increase the compound's ability to interact with viral particles. In all of the biflavonoids tested for antiviral activity by L i n et al, no compound has possessed as many hydroxyl groups as are present on this H. splendens biflavonoid. 5',3'"-Dihydroxyrobustaflavone possess a 5'-6 linkage between its monomeric units. This type of linkage is somewhat similar to the 3'-6 linkage of robustaflavone as the second subunits are both attached at the 6 position. As a result, the cyclic ether of the th  second subunits will have a more negative character than that of compounds such as amentoflavone which are bound at 8 position. Based on this comparison, it seems th  likely that 5',3'"-dihydroxyrobustaflavone might have a strong antiviral activity similar to that of robustaflavone.  3.4 Anti-viral Compounds from Iryanthera megistophylla A s a result of collaborations with Dr. Dong Sheng M i n g and Dr. Andres Lopez, newly isolated compounds from the Columbian vascular plant Iryanthera megistophylla became available for anti-PVX testing. Iryanthera megistophylla was one of several medicinal Columbian plants which was collected and studied by Dr. Andres Lopez as part of his doctoral degree. Nine compounds isolated from this plant by Dr. Dong Sheng M i n g were tested for P V X inhibition using the local lesion assay. The results of this, study are listed in Table 6 and have also been recently published in The Journal of Natural Products (Ming et al. 2002).  49  Table 6: A n t i - P V X activity of Iryanthera megistophylla compounds. Compounds were tested at 9.1mg/mL. Results have been published in M i n g et al. 2002. Quercetin and Ribavirin are positive controls.  Compound  Percent P V X Inhibition  1) Megislignan  0±60  2) Megislactone  30 ± 3 7  3) Grandinolide  30± 43  4) Iryantherin K  88± 18  5) Iryantherin L  67±39  6) Cinchonain lb  98±5  7) Cinchonain la  100±0  8) Procyanidin B-2  98±7  9) Cinchonain Ha  100±0  Quercetin  100±0  Ribavirin  94±7  The results of the antiviral testing in Table 6 indicate that the following five Iryanthera megistophylla compounds are highly antiviral against P V X : iryantherin K , cinchonain lb, cinchonain la, procyanidin B-2 and cinchonain Ila. The latter three compounds displayed an antiviral effect that was greater than the commonly used antiviral compound, ribavirin. The structures of these compounds are displayed along with their activities in Figure 16-Figure 22. A s is indicated by the structures, several of the compounds tested are diastereoisomers, or compounds with chiral centers which are not mirror images. Because of their different tree-dimensional shape, one would not necessarily expect these diastereoisomers to have the same level of antiviral activity. The two diastereoisomers iryantherin K & L differed in their degree of P V X inhibition  50  (88±18% and 67±39% respectively); however, the other diastereoisomers tested, cinchonain la & lb, exhibited nearly identical antiviral activities. When we compare the structures of the compounds with their antiviral activities in Figure 16-Figure 22, we see that megislignan, megislactone and grandinolide are the compounds with the least number of hydroxyl units and are also the least active of these compounds. A s has been mentioned earlier, in the case of the biflavonoids, a correlation has been noted with the amount of hydroxyl units and the antiviral activity (Lin, Anderson et al. 1997). O f the nine Iryanthera megistophylla compounds tested, only the six flavonoids compounds exhibited antiviral activity. Procyanidin B-2 and Cinchonain Ha are both biflavonoids and as a result of this study are the first to be published as having antiplantviral inhibition.  51  Figure 17: Procyanidin B-2 (98% inhibition)  OH  Figure 18: Megislactone  Ri  (30% inhibition)  Figure 19: Cinchonain OH  Cinchonain l b : R l = 3,4-dihydroxyphenyl, R 2 = H (98% inhibition) Cinchonain Ia:Rl = H , R 2 = 3,4-dihydroxyphenyl (100% inhibition)  Figure 21: Cinchonain Ha (100% inhibition)  Figure 22: Iryantherin Iryantherin K : R l = H , R 2 = 4-hydroxyphenyl (88% inhibition) Iryantherin L: R l = 4-hydroxyphenyl, R2 = H (67% inhibition)  3.5 Summary A large majority of the bryophyte species screened have been identified as exhibiting considerable antiviral activity against P V X and as a result, the bryophytes have been identified as a new source of antiviral activity. The bryophytes were originally selected for this study because it was thought that the biflavonoid content might confer antiviral activity; however, many of the active bryophyte species are from groups which are known not to contain flavonoids and thus we cannot conclude that biflavonoids are responsible for the antiviral activity of extracts from these organisms. A study of the chemistry of Hylocomium splendens was performed in order to gain a greater understanding of the cause of the antiviral activity o f the biflavonoid-containing mosses. While activity was observed in a fraction of the compounds which resemble the flavonoids components of the plant, attempts to isolate and identify these compounds were unsuccessful and as it stands, we do not know which compound(s) are responsible for the antiviral properties of these plants. Four biflavonoids were obtained for antiviral testing. O f these compounds, robustaflavone and hinokiflavone have been identified for the first time as being highly antiviral against P V X at very low concentrations. It is encouraging that these biflavonoids which were previously shown to be very effective at inhibiting several human viruses are now shown to be capable of inhibiting P V X . This implies that other compounds identified as being antiviral with the local lesion assay may also be effective against human viruses. In view of these results, it is also likely that other antiviral biflavonoids will contain activity against P V X .  53  Nine compounds from the Columbian plant Iryanthera megistophylla were for the first time tested for anti-PVX activity. O f these compounds the flavonids: iryanterin K , cinchonain lb, cinchonain l a and the biflavonoids: procyanidin B-2 and cinchonain Ha have been shown to be highly active against P V X . 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