Chemical investigation of a new endophytic fungus, Mycosphaerella sp. nov. strain F2140, associated with the foliage of the plant Psychotria horizontalis (Rubiaceae) in Panama, resulted in the isolation of cercosporin (1) and a new cercosporin analog (3) as the major components. The structures of minor compounds in the extract were elucidated by detailed spectroscopic analysis as 2-(2-butyl)-6-ethyl-3-hydroxy-6-methylcyclohex-2-ene-1, 5-dione (4), 3-(2-butyl)-6-ethyl-5-hydroxy-2-methoxy-6-methyl-cyclohex-2-enone (5), and an isomer of 5 (6). To study the influence of the hydroxy groups on the antiparasitic activity of cercosporin, compound 1 was acetylated to obtain derivative 2. The isolated compounds 1-6 were tested in vitro to determine their antiparasitic activity against the causal agents of malaria (Plasmodium falciparum), leishmaniasis (Leishmania donovani), and Chagas disease (Trypanosoma cruzi). Cytotoxicity and potential anticancer activity of these compounds were evaluated using mammalian Vero cells and MCF7 cancer cell lines, respectively. Compounds 1 and 2 displayed high potency against L. donovani (IC50 0.46 and 0.64 μM), T. cruzi (IC50 1.08 and 0.78 μM), P. falciparum (IC50 1.03 and 2.99 μM), and MCF7 cancer cell lines (IC50 4.68 and 3.56 μM). Compounds 3-6 were not active in these assays at a concentration of 10 μg/mL.
KursarT.A., Caballero-GeorgeC.G., CapsonT.L., Cubilla-RiosL., GerwickW.H., IbanezA., LiningtonR.G., McPhailK.L., Ortega-BarriaE., RomeroL.I., SolisP.N., ColeyP.D. (2006) Securing economic benefits and promoting conservation through bioprospecting. Bioscience, 56, 1005–1012. (b) Kursar TA, Caballero-George CC, Capson TL, Cubilla-Rios L, Gerwick WH, Heller MV, Ibanez A, Linington RG, McPhail KL, Ortega-Barria E, Romero LI, Coley PD. (2007) Linking bioprospecting with sustainable development and conservation: the Panama case. Biodiversity and Conservation, 16, 2789–2800. (c) Jimenez-RomeroC, Torres-MendozaD., Ureña-GonzalesL.D., Ortega-BarriaE., McPhailK.L., GerwickW.H., Cubilla-RiosL. (2007) Hydroxy-alkenylresorcinols from Stylogyne turbacensis. Journal of Natural Products, 70, 1249–1252. (d) Montenegro H, Gonzalez J, Ortega-Barria E, Cubilla-Rios L. (2007) Antiprotozoal activity of flavonoid glycosides isolated from Climedia sericea and Mosquitoxylon jamaicense. Pharmaceutical Biology, 45, 376–380. (e) Linington RG, Clark BR, Trimble EE, Almanza A, Ureña LD, Kyle DE, Gerwick WH, (2007) Antimalarial peptides from marine cyanobacteria: Isolation and structural elucidation of gallinamide A. Journal of Natural Products, 72, 14–17. (f) Medina RA, Goeger DE, Hills P, Mooberry SL, Huang N, Romero, LI, Ortega-Barria, E, Gerwick, WH, McPhail KL. (2008) Coibamide A, a potent antiproliferative cyclic depsipeptide from the Panamanian marine cyanobacterium Leptolyngbya sp. Journal of American Chemical Society, 130, 6324–6325. (g) Jimenez-Romero C, Ortega-Barria E, Arnold AE, Cubilla-Rios L. (2008) Activity against Plasmodium falciparum of lactones isolated from the endophytic fungus Xylaria sp. Pharmaceutical Biology, 46, 700–703. (h) Martinez-Luis S, Della-Tonga G, Coley PD, Kursar TA, Gerwick WH, Cubilla-Rios L. (2008) Antileishmanial constituents of the Panamanian endophytic fungus Edenia sp. Journal of Natural Products, 71, 2011–2014.
2.
KuyamaS., TamuraT. (1957) Cercosporin. A pigment of cercosporina kikuchii matsumoto et tomoyasu. i. Cultivation of fungus, isolation and purification of pigment. Journal of American Chemical Society, 79, 5725–5726. (b) Assante G, Locci R, Camarda L, Merlini L, Nasini G. (1977) Screening of the genus Cercospora for secondary metabolites. Phytochemistry, 16, 243–247. (c) Kuyama S, Tamura T. (1957) Cercosporin. A pigment of cercosporina kikuchii matsumoto et tomoyasu. ii. Physical and chemical properties of cercosporin and its derivatives. Jouranl of American Chemical Society, 79, 5726–5729. (d) Yamazaki S, Ogawa T. (1972) The chemistry and stereochemistry of cercosporin. Agricultural and Biological Chemistry, 36, 1707-1718.
3.
BringmannG., GuntherC., OchseM., SchuppO., TaslerS. (2001) Axially chiral biaryls, a multi-facetted class of stereochemically, biosynthetically, and pharmacologically intriguing secondary metabolites. Progress in Chemistry of Organic Natural Products, 82, 1–249.
4.
DaubM.E. (1982) Cercosporin, a photosensitizing toxin from Cercospora species. Phytopathology, 72, 370–374.
5.
DaubM.E., (1982) Peroxidation of tobacco membrane lipids by the photosensitizing toxin, cercosporin. Plant Physiology, 69, 1361–1364.
6.
GoodwinS.B., DunkleL.D., ZismannV.L. (2001) Phylogenetic analysis of Cercospora and Mycosphaerella based on the internal transcribed spacer region of ribosomal DNA. Phytopathology, 91, 648–658.
7.
ArnoldA.E., HenkD.A., EellsR.A., LutzoniF., VilgalysR. (2007) Diversity and phylogenetic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR. Mycologia, 99, 185–206. (b) Arnold AE, Miadlikowska J, Higgins KL, Sarvate SD, Gugger P, Way A, Hofstetter V, Kauff, F, Lutzoni F. (2009) Hyperdiverse fungal endophytes and endolichenic fungi elucidate the evolution of major ecological modes in the Ascomycota. Systematic Biology, 58, 283–297. (c) Arnold AE, Gunatilaka MK, Varughese T, Moreno E, Coley PD, Kursar TA, Cubilla LC. (In preparation) A novel species of Mycosphaerella endophytic in foliage of Psychotria horizontalis (Rubiaceae) in Panama. To be submitted to Fungal Diversity, 2011.
8.
MorganB.J., DeyS., JohnsonS.W., KozlowskiM.C. (2009) Total synthesis of cercosporin and new photodynamic perylenequinones: Inhibition of the protein kinase C regulatory domain. Journal of American Chemical Society, 131, 9413–9425.
9.
CorreaJ.E., RiosC.H., CastilloA.R., RomeroL.I., Ortega-BarriraE., ColeyP.D., KursarT.A., HellerM.H., GerwickW H., Cubilla-RiosL. (2006) Minor alkaloids from Guatteria dumentorum with antileishmanial activity. Planta Medica, 72, 270–272.
10.
ArnoldA.E., LutzoniF. (2007) Diversity and host range of foliar fungal endophytes: Are tropical leaves biodiversity hotspots?Ecology, 88, 541–549.
11.
WhiteT.J., BrunsT., LeeS., TaylorJ. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In InnisM.A., GelfandD.H., SninskyJ.J., WhiteT.J., (Ed) PCR protocols: A guide to methods and applications.San Diego, Academic Press, pp. 222–315.
12.
HoffmanM., ArnoldA.E. (2008) Geography and host identity interact to shape communities of endophytic fungi in cupressaceous trees. Mycological Research, 112, 331–344.
13.
HigginsK.L., ArnoldA.E., MiadlikowskaJ., SarvateS.D., LutzoniF. (2007) Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three major plant lineages. Molecular Phylogenetics and Evolution, 42, 543–555.
14.
EwingB., HillierL., WendlM., GreenP. (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Research, 8, 175–185; (b) Ewing B, Green P. (1998) Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Research, 8, 186–194.
15.
CalderonA., RomeroL.I., Ortega-BarriaE., BrunR., CorreaM.A., GuptaM.P. (2006) Evaluation of larvicidal and in vitro antiparasitic activities of plants in a biodiversity plot in the Altos de Campana National Park, Panama. Pharmaceutical Biology, 44, 1–16.
16.
BucknerF.S., VerlindeCLMJ, La FlammeA.C., Van VoorhisW.C. (1996) Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrobial Agents and Chemotherapy, 40, 2592–2597.
17.
SánchezL.A., CapitanZ., RomeroL.I., Ortega-BarríaE., GerwickW.H., Cubilla-RiosL. (2007) Bio-Assay guided isolation of germacranes with anti-protozoan activity from Magnolia sororum. Natural Product Communications, 2, 1065–1198.
18.
TragerW., JensenJ.B. (1976) Human malaria parasites in continuous culture. Science, 193, 673–675. (b) Corbett Y, Herrera L, Gonzalez J, Cubilla L, Capson TL, Coley PD, Kursar TA, Romero LI, Ortega-Barria E. (2004) A Novel DNA-Based Microfluorimetric Method to Evaluate Antimalarial Drug Activity. American Journal of Tropical Medicine and Hygiene, 70, 119–124.
19.
MonksA., ScudieroD., SkehanP., ShoemakerR., PaullK., VisticaD., HoseC., LangleyJ., CroniseP., Vaigro-WolffA., Gray-GoodrichM., CampbellH., MayoJ., BoydM. (1991) Feasibility of a High-Flux Anticancer Drug Screen Using a Diverse Panel of Cultured Human Tumor Cell Lines. Journal of National Cancer Institute, 83, 757–766.
20.
Van de LoosdrechtA.A., BeelenR.H.J., OssenkoppeleG.J., BroekhovenM.G., LangenhuijsenM.M. (1994) A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. Journal of Immunological Methods, 174, 311–320.
