The Occurrence of Photosensitizers Among Higher Plants - ACS

Chapter 11

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The Occurrence of Photosensitizers Among Higher Plants Kelsey R. Downum and Jinghai Wen Department of Biological Sciences, Florida International University, Miami, FL 33199 and Fairchild Tropical Garden, 10901 Old Cutler Road, Miami, FL 33156

More than 100 photosensitizers or phototoxins have been identified from higher plant tissues. These light-activated metabolites are broad-spectrum biocidal agents which have been implicated in a variety of plant defensive responses. The phototoxic metabolites isolated to date belong to 15 different phytochemical classes and are products of at least four biosynthetic pathways - fatty acid, polyketide, shikimate and terpenoid. A review of the phytochemical literature confirms the presence of phototoxic components in 35 families. Using disk-diffusion antimicrobial bioassays to survey higher plants for phototoxic activity, we have found light-activated bioactivity in extracts of taxa belonging to a variety of taxonomically disparate families including the: Acanthaceae, Campanulaceae, Gesnariaceae, Loganiaceae, Malpigiaceae, Papaveraceae, Phytolaccaceae, Piperaceae and Sapotaceae. This brings the total number of families that have been found to contain photosensitizers or exhibit phototoxic activity to 44.

Botanical phototoxins or photosensitizers are a structurally-diverse assemblage of plant metabolites that are grouped together because they mediate similar biological actions; namely, they catalyze toxic reactions following the absorption of light energy (7). These reactions are often lethal toward organisms that compete with or are otherwise harmful to plants, including pathogens, parasites, herbivores and other plants (7). Since the mechanisms of action, cellular targets and organismal effects of a variety of plant photosensitizers are reviewed elsewhere (7-3), these issues will not be dealt with in the present paper. Instead, an overview of the current state of knowledge regarding the phytochemical diversity, biosynthetic origins and taxonomic occurrence of photosensitizers among flowering plant families will be presented.

0097-6156/95/0616-0135$12.00/0 © 1995 American Chemical Society In Light-Activated Pest Control; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Sesquiterpene

Extended Quinone

Fig. 2: Phototoxins derived from products of other biochemical pathways

In Light-Activated Pest Control; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.

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Biosynthetic Origins More than 100 phototoxic natural products have been identified since their discovery in higher plants slightly more than 20 years ago. Compoundsfromat least 15 different phytochemical classes are known to enter a "phototoxic" state following the absorption of light energy. Molecules that share this type of biological activity have diverse biochemical origins and are derived from four distinct metabolic pathways - the shikimate, fatty acid, terpenoid and polyketide pathways. The biosynthetic origins of phototoxins identified to date are summarized in Figures 1 & 2. Photosensitizers derived from the shikimic acid pathway, the major biochemical pathway that gives rise to most of the phototoxic metabolites, are shown in Figure 1. Aromatic amino acids produced by this pathway are precursors for many of the polycyclic, aromatic photosensitizers. Phenylalanine serves as the starting molecule from which various coumarin, furanocoumarin, lignan and pterocarpan phototoxins are built. Isoquinoline and benzophenanthrene phototoxins are derived from tyrosine, while βcarboline and quinoline photosensitizers resultfromstructural modifications to tryptophan. Anthranilic acid, another product of the shikimate pathway, givesriseto furanoquinolinetype photosensitizers. The fatty acid, terpenoid, and polyketide pathways giveriseto a variety of essential higher plant metabolites as well as to other photosensitizer types (Fig. 2). Linear polyacetylenes, thiophenes and various otherring-stabilizedpolyynes are formed from C fatty acid precursors following shortening and desaturation of the hydrocarbon chain (4). Terpenoids, the phytochemical class most recently shown to mediate phototoxic reactions (5-8), are formed by the condensation and modification of isoprene (C ) units. Enzymes of the polyketide pathway are responsible for the conversion of 2-carbon acetate units into dictamnine (a furanochromone), extended quinones like hypericin and the benzyl functions of acetophenones. Furanocoumarins and acetophenones are somewhat unique among plant photosensitizers in that they require the coordinated action of two biosynthetic pathways for theirfinalstructures, i.e., they have mixed biosynthetic origins. The furan ring of furanocoumarins and the pyran ring of acetophenones are formed by the addition of isoprene units to the coumaryl portion of furanocumarins (formed via shikimate pathway) and the benzyl moiety of acetophenone derivatives (formed via the polyketide pathway). 18

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Taxonomic Occurrence A survey of the literature reveals that a substantial number of flowering-plant families contain phototoxic constituents. Phytochemical studies have confirmed the presence of derivatives from one or more classes of photosensitizers discussed above in at least 35 families (Tables I & II); bioassay studies suggest their presence in an even greater number of families. Twelve plant families have taxa that make multiple types of photosensitizers (Table I). Members of the Rutaceae (citrus family) are of particular note because of their ability to synthesize the broadest array of photosensitizers with specific examples reported in the literature from the β-carbolines, coumarins, furanocoumarins, furanoquinolines, isoquinolines and sesquiterpenes. Acetophenone, acetylene, thiophene and furanocoumarin



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Table I. Flowering plant families with two or more classes of photosensitizers. FAMILY PHYTOCHEMICAL CLASS REFERENCE 9 Coumarins, Furanochromones, Furanocoumarins Apiaceae 4,10,11 Acetophenones, Acetylenes, Furanocoumarins, Asteraceae Thiophenes 9,12 Benzophenanthrenes, Furanocoumarins Dipsacaceae 9,13, 18,19 β-Carbolines, Furanocoumarins, Pterocarpans Fabaceae 9,13,17 β-Carbolines, Extended Quinones, Furanocoumarins Fagaceae 9,14 Coumarins, Furanocoumarins Moraceae 12 Benzophenanthrenes, Isoquinolines Papaveraceae 9,13 β-Carbolines, Coumarins Polygonaceae 12,13 β-Carbolines, Isoquinolines, Quinolines Rubiaceae 5,6,9,12,13 β-Carbolines, Coumarins, Furanocoumarins, Rutaceae Furanoquinolines, Isoquinolines, Sesquiterpenes 9,13 β-Carbolines, Furanocoumarins Solanaceae 13,16 β-Carbolines, Lignans Zygophyllaceae

Table Π. Flowering plant families from which one class of photosensitizer has been reported. PHYTOCHEMICAL FAMILIES REFERENCE CLASS 9 Apocyanaceae, Bignoniaceae, β-Carbolines Calycanthaceae, Chenopodiaceae, Combretaceae, Cyperaceae, Malpigiaceae, Passifloraceae 12 Sapindaceae Benzophenanthrenes 12 Annonaceae, Berberidaceae, Euphorbiaceae, Isoquinolines Juglandaceae, Magnoliaceae, Menispermaceae, Ranunculaceae 9 Amaranthaceae, Orchidaceae, Pittosporaceae Furanocoumarins 17 Hypericaceae Extended Quinones 7,8 Malvaceae Sesquiterpenes 12 Nyssaceae Quinolines



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photosensitizers occur in various tribes of the Asteraceae (sunflower family). β-Carbolines and furanocoumarins have the widest occurrence among other families that synthesize multiple types of photosensitizers. Taxa belonging to 23 other families contain metabolitesfromat least one of the phototoxic phytochemical classes (Table II). β-Carboline and isoquinoline derivatives occur in the majority of these families with benzophenanthrenes, furanocoumarins, extended quinones, sesquiterpenes and quinolines occurring in various other families. Little work has been done to document the structural variation and biological activity of phototoxins from these plants. Further study is necessary to identify the specific phytochemicals responsible for phototoxicity and to determine the scope of organisms susceptible to their detrimental effects. The phytochemical diversity, albeit patchy occurrence, of phototoxic phyto chemicals among higher plants, makes chromatographic methods of screening botanical tissues for specific types of photosensitizers of limited use. Instead, a rapid and sensitive antimicrobial bioassay can be used to survey large numbers of plants for phototoxic activity (14). Since a range of organisms (e.g., pathogens, insect herbivores, etc.) are sensitive to the toxic action of most plant photosensitizers (20), antimicrobial bioassays offer an effective method of screening plants for phototoxic metabolites that can potentially affect a wide variety of organisms. For the past 10 years, my laboratory has employed diskdiffusion antimicrobial bioassays to survey neotropical plants for phototoxic activity. We have bioassayed nearly 2,000 plant species from 250 genera and ca. 80 families. Methanolic extracts offreshplant tissue either field collected in Mexico, Costa Rica or Brazil, or obtained from the living collections at Fairchild Tropical Garden or the USDA Plant Induction Station in Miami, FL were bioassayed using established methodology (20). Extracts from approximately 5% of the species bioassayed tested positive for phototoxins (Table ΙΠ). These plants belong to 16 families - four of which had previously been shown to have photosensitizer-containing species (Asteraceae, Hypericaceae, Moraceae and Rutaceae). HPLC analysis of extractsfromphotosensitizer-containing plants revealed the presence of compounds with UV absorption spectra characteristic of acetylenic and thiophenic components (Asteraceae), extended quinones (Hypericaceae), and coumarins/furanocoumarins (Moraceae and Rutaceae) (14-15, 28 and unpublished results). Nine of the families listed in Table III were not previously known to produce phototoxic phytochemicals. Indication of phototoxic components in extracts from plants belonging to the Acanthaceae, Campanulaceae, Gesnariaceae, Loganiaceae, Malpigiaceae, Papaveraceae, Phytolaccaceae, Piperaceae and Sapotaceae represent important new findings. Preliminary examination of the extracts by reverse-phase HPLC suggest that the phototoxic agents in these plants do not absorb in spectral regions characteristic of any of the known phototoxin classes. We have become particularly interested in identifying the phototoxic constituents from the genus Piper (Piperaceae) because of the prevalence of phototoxins in leaf extracts (30 of 33 species were phototoxic against Bacillus cereus) and the ecological importance of the genus in the New World tropics. Efforts to isolate the UV-activated constituent(s) from various Piper extracts using bioassay-directed fractionation of extracted leaf material have been unsuccessful as phototoxicity disappears during thefractionationprocedure. It is not yet clear whether the loss of bioactivity is due to chemical degradation or the result



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Table III. Tropical/subtropical plants that have tested positive for phototoxic components using the disk difussion bioassay for antimicrobial activity (20). Numbers in parentheses represent the number of species that were phototoxic. PHOTOTOXIC GENERA FAMILIES Ambrosia (1), Coreopsis (1), Dyssodia (2), Asteraceae Eclipta (1), Erigeron (1), Flaveria (4), Gaillardia (1), Rudbeckia (1), Tithonia (1) Acanthaceae* Herpetacanthus (1) Campanulaceae* Hippobroma (1) Gesnariaceae* Bresera (1), Palicoria (1) Hypericum (2) Hypericaceae Loganiaceae* Spigelia (1) Malpigaceae* Banisteriopsis (2), Marscagnia (1) Clarisia, Dorstenia (5), Fatoua (1), Ficus (2), Tropis () Moraceae Papaveraceae* Argemone (1) Phytolaccaceae* Phytolacca (1) Piperaceae* Piper (33), Pothomorphe (1) Rubiaceae Psycotria (2), Cephalis (4) Afraegle (1), Atalantia (1), Citrus (10), Microcitrus (1), Rutaceae Severinia (1), Swinglea (1) Sapotaceae* Pouteria (1) Solanaceae Solanum (1), Witheringia (2) *Represents a new report of photosensitizers/phototoxic activity in this plant family.

of physical separation of extract components during the isolation procedure. We suspect the latter, since HPLC analysis of fraction components does not suggest chemical degradation. A range of phytochemicals have been reportedfromPiper that could be responsible for the phototoxic action of extracts - these include various acetophenones (27), alkaloids (22-24), lignans (25-26) and terpenoids (27). In an effort to test some of these compounds, samples of dillapiole (a methylene dioxyphenylpropene) and three lignans isolated from P. decurrans were obtained from Dr. Thor Arnason's laboratory (Univ. of Ottawa) for antimicrobial bioassay. Although these compounds mediated varying degrees of antimicrobial activity toward B. cereus, they were not responsible for the phototoxic action observed with crude plant extracts (Table IV). Efforts to isolate the phototoxic compounds from Piper extracts are ongoing. Survey studies to bioassay new plant extracts for phototoxic antimicrobial activity continue in an effort to establish the prevalence of phototoxic phytochemicals among higher plants, as do phytochemical studies to isolate and identify new phototoxic components that are found in our surveys. Ninety-five percent of the plants examined thus far have tested negative for phototoxic activity. It is not practical to list all of the genera that lacked phototoxic activity here. Plant families lacking phototoxicity have been identified in previous publications (7, 28). A listing of the genera and species that have been tested is maintained and can be provided upon request.


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Occurrence ofPhotosensitizers Among Higher Plants 14

Table IV. Antimicrobial activity of lignans from Piper decurrens against Bacillus cereus. NAME

STRUCTURES

INHIBITION ZONE * Dark UVA


7.8 ±0.47 **

9.7 ±0.85

7.7 ± 0 . 6 4

* Inhibition zones expressed in mm ± standard deviation; (-) indicates the absence of an inhibition zone. All lignans were bioassayed with doses of 10 μg/disk; dillapiole was bioassayed at a dose of 0.8 mg/disk. ** The data were analysed using ANOVA and no significant differences were detected between dark and UVA treatment at p=0.05.

Conclusion Phytochemical and bioassay studies have identified a large number of photosensitizers and phototoxin-containing plant species since their initial discovery more than two decades ago. Studies of the potential applications of several types of plant-derived photosensitizers as biocontrol agents are ongoing in a number of laboratories in Canada and the U.S. (see following chapters). To date, fewer than 100 higher plant families (ca. 30% of the extant families) have been analyzed for phototoxins or phototoxic activity. With the exception of a few families (e.g., Apiaceae, Asteraceae, Moraceae, and Rutaceae), only a small fraction of the species in each family has been examined. Since phototoxins or phototoxic activity seem to occur in taxa belonging to at least 10-15% of the higher plant families, it is reasonable to suspect that many more phototoxic phytochemicals remain to be discovered, and that additional families of plants will be found to contain light-activated chemicals.


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Continued exploratory research will help to: i) clarify the taxonomic relationships between plants that contain phototoxic phytochemicals; ii) assess the prevalence of lightactivated defenses among higher plants; and iii) provide new photosensitizers with potential as biocontrol agents. Acknowledgments. The authors would like to thank the following individuals for providing lignan standards - Denise Schauaret, Thor Arnason and Tony Durst (University of Ottawa) and identification of various plant materials - Luis Poveda and Pablo Sanchez (Universidad Nacional de Costa Rica). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17. 18. 19. 20. 21. 22. 23.

Downum, K.R. NewPhytol.1992, 122, 401-420. Towers, G.H.N. Can. J. Bot. 1984, 62, 900-2911. Downum, K.R.; Rodriguez, E. J. Chem.Ecol.1986, 823-834. Bohlmann, F.; Burkhardt, T.; Zdero, C. Naturally Occurring Acetylenes. Academic Press, London, 1973. Asthana, Α.; Larson, R.A.; Marley, K.A.; Tuveson, R.W. Photochem. Photobiol. 1992, 56, 211-222. Green, E.S.; Berenbaum, M.R. Photochem.Photobiol.1994, 60, 459. Sun, T.J.; Melcher, U; Essenberg, M.Physiol.Mol.Plant Path. 1988, 33, 115-136. Sun, T.J.; Essenberg, M; Melcher, U. Mol. Plant-Microbe Interact., 1989, 2, 139147. Murray, R.D.H.; Mendez, J.; Brown, S.A. The Natural Coumarins: Occurrence, Chemistry and Biochemistry. J. Wiley & Sons Ltd., Chichester, 1982. Miyakado, M.; Ohno, N.; Yoshiokka, H.; Mabry, T.J. Phytochem. 1978, 17, 143144. Proksch, P.; Rodriguez, E. Phytochem. 1983, 22, 2335-2348. Raffauf, R.F. A Handbook of Alkaloids and Alkaloid-Containing Plants. J. Wiley & Sons, New York, NY, 1970. Allen, J.R.F.; Holmstedt, B.R. Phytochem. 1980, 19, 1573-1582. Swain, L.A.; Downum, K.R. Biochem. Syst.Ecol.1990, 18, 153-156. Downum, K.R.; Provost, D.; Swain, L. In Bioactive Molecules: Chemistry and Biology of Naturally-Occurring Acetylenes and Related Compounds (NOA Lam, J; Breteler, H.; Arnason, T.; Hansen, L., Eds.; Elsevier: Amsterdam, 1988, Vol. 7; pp. 151-158. MacRae, W.D.; Towers, G.H.N. Phytochem. 1984, 23, 1207-1221. Thompson, R.H. Naturally Occurring Quinones; Academic Press, London, 1971. Robeson, D.J.; Harborne, J.B. Phytochem. 1980, 2359-2365. Smith, D.A.; Banks, S.W. Phytochem. 1986, 25, 979-995. Downum, K.R.; Swain, L.A.; Faleiro, L.J. Arch. Insect Biochem.Physiol.1991, 17, 201-211. Diaz D., P.P.; Arias C., T; Joseph-Nathan, P.J. Phytochem. 1987, 26, 809-811. Shah, S.; Kalla, A.K.; Dhar, K.L. Phytochem. 1986, 25 1997-1998. Duh, C-Y.; Wu, Y-C; Wank, S-K. Phytochem. 1990, 29, 2689-2691.


11. DOWNUM & WEN 24. 25. 26.


27. 28.


Dominguez, X.A.; Verde S., J.; Sucar, S.; Trevino, R. Phytochem. 1986, 25, 239240. Badheka, L.P.; Prabhu, B.R.; Mulchandani, N.B. Phytochem. 1987, 26, 2033-2036. Koul, S.K.; Taneja, S.C.; Pushpangadan, P.; Dhar, K.L. Phytochem. 1988, 27, 1479-1482. Russell, G.R.; Jennings, W.G. J. Agr. Food Chem. 1969, 17, 1107-1112. Swain, L. Α.; Downum, K.R. In Naturally Occurring Pest Bioregulators; Hedin, P.A., Ed.; ACS Symp. Ser. No. 449; ACS, Washington, D.C., 1991; pp. 361-370.

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The Occurrence of Photosensitizers Among Higher Plants - ACS

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