Antimicrobial Secondary Metabolites from Endophytic Fungi of Goldenseal (Hydrastis canadensis) Joseph Egan,1 Amninder Kaur,1 Adam R. Brown,1 Huzefa Raja,1 Nicholas H. Oberlies,1 Nadja B. Cech1 1Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402 INTRODUCTION ANTIMICROBIAL SECONDARY METABOLITES SECONDARY METABOLITES WITH ALTERNATIVE BIOACTIVITIES Spiciferone A Piperazinedione Cordypyridone B Dihydrospiciferol A 10,11-Dehydrocurvularin Altenuene Macrosphelide A Equisetin CONCLUSIONS The antimicrobial secondary metabolites isolated from the endophytic fungal extracts support the initial hypothesis that these fungi and the secondary metabolites they produce may contribute to the overall antimicrobial activity of plant. Although berberine derived from the plant extract can be responsible for some antimicrobial activities, the results presented here suggest that the role of endophytic fungi inhabiting these plants snot be underestimated. Goldenseal (Hydrastis canadensis L. Ranunculaceae), a plant native to the Appalachian mountains of the United States, is used in traditional medicine to treat topical infections. Previous studies have demonstrated antimicrobial activity for goldenseal’s major secondary metabolite, berberine. However, it was hypothesized that compounds produced by endophytic fungi might also play an important role in its antimicrobial activity. Approximately seventy fungal endophytes were isolated and identified from H. canadensis and crude fungal extracts were tested for antimicrobial activity against Staphylococcus aureus. Major metabolites were isolated from several of these fungi, structures elucidated by NMR and MS, and biological activities evaluated via MIC assays. FUTURE DIRECTIONS The search for new antimicrobial agents has become an ever expanding field due to the development of antimicrobial resistance. The pursuit of new sources of antibiotics or suppliers of existing antibiotics encourages the investigation of new natural product sources such as endophytic fungi. Bioactivity guided fractionation using whole cell phenotypic assays includes certain variables that are not prevalent in targeted assays, leading to potential alternative therapeutic options that may otherwise be overlooked. Future experiments will be targeted towards addressing the biological relevance of compounds beyond MIC calculations and taking into account variables such as in vivo solubility and synergistic small molecule interactions. METHODS Figure 1. Percent Growth inhibition of S. aureus by crude extracts of H. canadensis Antimicrobial assay Antimicrobial micro dilution assays against S. aureus were carried out on the crude extracts at 200 µg/mL and 20 µg/mL concentrations. (Figure 1) MIC assays were conducted on pure compounds (purity >95%). (Figure 2) Isolation and Identification of Secondary Metabolites Fungal isolates were collected, extracted, and fractionated using flash chromatography and High Pressure Liquid Chromatography (HPLC). Pure compounds were analyzed using ESI-Q-TOF and LC-Orbitrap mass spectroscopy as well as NMR spectroscopy . REFERENCES 1.Scazzocchio, F.; Cometa, M. F.; Tomassini, L.; Palmery, M. Planta Med. 2001, 67, 561-564. 2.Amin, A. H.; Subbaiah, T. V.; Abbasi, K. M. Canadian Journal of Microbiology 1969, 15, 1067-&. 3.Hayashi, M.; Kim, Y. P.; Hiraoka, H.; Natori, M.; Takamatsu, S.; Kawakubo, T.; Masuma, R.; Komiyama, K.; Omura, S. Journal of Antibiotics 1995, 48, 1435-1439. 4.Kjer, J. Ph.D Dissertation, Heinrich-Heine Universität Düsseldorf, 2010. 5.Burmeist.Hr; Bennett, G. A.; Vesonder, R. F.; Hesselti.Cw Antimicrobial Agents and Chemotherapy 1974, 5, 634-639. 6.(a) Nakajima, H.; Fujimoto, H.; Kimura, Y.; Hamasaki, T. Biosci., Biotechnol., Biochem. 1993, 57, 1938-9; (b) Nakajima, H.; Fukuyama, K.; Fujimoto, H.; Baba, T.; Hamasaki, T. J. Chem. Soc., Perkin Trans. 1 1994, 1865-9. RESULTS Of the 70 extracts of endophytic fungi isolated from H. canadensis, at least 12 isolates were found to exhibit antimicrobial properties in susceptibility assays against S. aureus. Fungal isolates from H. canadensis were grown on rice and identified using DNA analysis. Chemical separation of a few selected extracts resulted in the isolation of 4 known antimicrobial compounds that were produced as major secondary metabolites. Macrosphelide A was isolated in large quantities from an endophytic isolate of Pyrenochaeta cava3 (Figure 3). Other known antimicrobial agents such as altenuene4 and equisetin5 were also encountered from other fungal isolates. MIC assays were run on pure compounds to determine inhibitory concentrations. Some extracts that proved to be antimicrobial in their crude extract did not yield pure compounds in enough quantity to elucidate the structure of the major metabolite. ACKNOWLEDGMENTS REPRESENTATIVE NMR SPECTRUM OF DIHYDROSPICIFEROL A Thanks to Daniel Todd and the rest of Cech-lab for their continued support, be it technical, moral, and/or academic. Thanks to Dr. Franklin J. Moy and Dr. Brandie M. Ehrmann for training and utilization of their respective spectroscopy laboratories. Special thanks to Dr. Nadja Cech for support, training, and guidance. This research was supported by R01 AT006860 from the National Center for Complementary and Alternative Medicine/National Institutes of Health, Bethesda, MD, USA. Additional funding was provided by the North Carolina Biotechnology Center (2011-BRG-1206). Figure 2. Determination of MIC for equisetin H-7 Hβ-9 H2-5 H2-6 and Hα-9 H3-10 8-CH3 3-CH3 2-CH3 Figure 3. Pyrenochaeta cava grown on rice medium (left), and extraction of crude extract (right)