1. TEMPLATE DESIGN © 2008 www.PosterPresentations.com Assessment of extracts prepared from native St. John’s wort species ( Hypericum spp.) on anxiety/depression-related behaviors and neural pathways in zebrafish ( Danio rerio ) Jeremy Duke, Katie Ichikawa, Julian Pittman, Michael Wayne Morris, Shaoyang Liu Department of Biological and Environmental Sciences, Troy University, Troy, AL 36082 Introduction Phytomedicines based on extracts from the herb Hypericum perforatum (St. John’s wort) are often referenced to with the moniker “nature’s Prozac”. Although St. John's wort has been known for centuries and has been used for a variety of medicinal purposes, understanding of its activity and mechanisms-of-action is not well understood. Several compounds present in Hypericum perforatum are unique in that they are reported to impact a wide range of neurotransmitters directly, or indirectly, through receptor sensitivity and regulation. It is hypothesized in this study that other Hypericum species are sources of some of the same bioactive compounds that are also in Hypericum perforatum. Zebrafish are becoming a commonly used alternative experimental organism in biomedical research because the physiology and morphology of this species are homologous to humans. Furthermore, the ease of drug exposure with this taxon and subsequent clear behavioral changes make this organism a powerful model to investigate the neural substrates of anxiety/depression. The aims of this study were to determine presence of known Hypericum perforatum bioactive compounds in native Hypericum species, and analyze efficacy for anxiety/depression-related behaviors following exposure to Hypericum species extracts in zebrafish (Danio rerio). MethodsResults Conclusions References Native St. John’s wort (Hypericum spp.) are promising as sources of antidepressant-like compounds and possible neuroactive pharmaceutical lead structures. Therefore, further investigations of the bioactive compounds in Hypericum perforatum and Hypericum species native to the southeastern United States for additional secondary plant metabolites with potential antidepressant properties are needed. Employing behavioral measures (novel tank diving test) (Fig. 3), Hypericum sp. extract effects were compared to typical anxiolytics and antidepressants: fluoxetine (Prozac®) 100 µg/L, buspirone (Buspar®) 25mg/L, diazepam (Valium) 25mg/L. Figure 3. Schematic of pre-treatment dosage administration and behavioral analysis (novel tank diving test). Figure 4. Behavioral effects of chronic intermittent exposure to 3% ethanol daily for 2 weeks, subsequent withdrawal, followed by buspirone, diazepam and fluoxetine exposure for 2 weeks. Barnes, J., Anderson, L. A., Phillipson, J. D. 2001. St. John’s wort (Hypericum perforatum L.): a review of its chemistry, pharmacology and clinical properties. Journal of Pharmacy and Pharmacology 53, 583-600. Bennett, D.A. (Jr.), Phun, L., Polk, J. F., Voglino, S. A., Zlotnik, V., Raffa, R. 1998. Neuropharmacology of St. John’s Wort. Annals Pharmacother 32 (11),1201-1208. Best, J. 2004. Zebrafish: An in vivo model for the study of neurological diseases. Neuropsychiatric Disease and Treatment. 4 (3), 567-576. Caccia, S., Gobbi M., 2009. St. John’s wort components and the brain: uptake, concentrations reached and the mechanisms underlying pharmacological effects. Current Drug Metabolism 10, 1055-1065. Crockett, S., Schaueber, B., Khan, I.A. 2005. Phytochemical profiling of New and Old World Hypericum (St. John’s wort) species. Phytochemical Analysis 16(6):478-485. Filippini, R., Piovani, A., Borsarini, A., Caniato, R. 2010. Study of dynamic accumulation of secondary metabolites in three subspecies of Hypericum perforatum. Fitoterapia 81:115-119. Griffith, T.N., Varela-Nallar, L., Dinamarca, M.C., Inestrosa, N.C. 2010. Neurobiological effects of hyperforin and its potential in Alzheimer's disease therapy. Current Medicinal Chemistry 17:391-406. Kasper, S., 2001. Hypericum perforatum – a review of clinical studies. Pharmacopsychiatry 34 (Suppl), S51-S55. Kasper, S., Caraci, F., Forti, B., Drago, F., Aguglia, E. 2010. Efficacy and tolerability of Hypericum extract for the treatment of mild to moderate depression. European Neuropsychopharmacology 20:747-765. Wurglics, M., Schubert-Zsilavecz, M. 2006. Hypericum perforatum: a ‘modern’ herbal antidepressant: Pharmacokinetics of active ingredients. Clin Pharmacokinet 45 (5), 449-468. Acknowledgements Figure 5. Behavioral effects of chronic intermittent exposure to 3% ethanol daily for 2 weeks, followed by extract exposure (10%) for 2 weeks. This work was supported by a Tri-Beta Grant, the Department of Biological and Environmental Sciences, Troy University, and by a Faculty Development Research Grant awarded to JP and MWM. HPLC analysis of native Hypericum species shows presence of medicinal phytochemicals also occurring in Hypericum perforatum (Fig. 7). Several species of St. John’s wort (Hypericum spp.) native to the southeastern United States (H. fasciculatum, H. microsepalum, H. punctatum, H. cistifolium, and H. prolificum) are being investigated as potential sources of chemical compounds with antidepressant properties that are already well known in the Eurasian H. perforatum. Hypericin, hyperforin, rutin, and quercetin are among the psychiatrically beneficial compounds previously identified in H. perforatum that could hypothetically be present in American species of Hypericum. The purpose of this study is to test for possible antidepressant effects of extracts prepared from Hypericum species in zebrafish (Danio rerio). Extracts were prepared from dried material of native St. John’s wort species collected at anthesis and subsequently administered to zebrafish, which were pharmacologically- induced to model depression. Preliminary test results show positive behavioral effects using a Novel Tank Diving Test, and detection of bioactive compounds was conducted through the use of High Performance Liquid Chromatography (HPLC). Changes in dopamine, serotonin, and norepinephrine levels are currently being investigated using an enzyme-linked immunosorbent assay (ELISA). The preliminary results from this study highlight the potential efficacy of species of St. John’s wort (Hypericum spp.) commonly found in the southeastern United States. Abstract Material of five native Hypericum species, including Hypericum microsepalum (Fig. 1) and Hypericum punctatum (Fig. 2), was collected at anthesis and dessicated at room temperature in the dark to preserve light-sensitive phytochemicals. Subsequently, the plant material was ground and compounds liquid extracted with methanol and dissolved into DMSO before being brought to 3% dosage volume. Figure 1. Hypericum microsepalum showing flowers, leaves, and fruits before collection. Figure 6. Comparison of behavioral effects of chronic intermittent exposure to 3% ethanol daily for 2 weeks, followed by Hypericum extract exposure (3%) for (A) 1 week and (B) 2 weeks. A.A.B.B. HPLC was performed using a C18 column, through which a mobile phase of purified water with 0.1% formic acid and acetonitrile with 0.1% formic acid was used. Figure 2. Concentrated regions of hypericin are found in the punctate dots of Hypericum punctatum. Improvements in behavior similar to that of typical anxiolytics and antidepressants (Figs. 4 & 5) were observed in zebrafish treated with extracts of native Hypericum species (Fig. 6). A.B.B. C.D.D. Figure 7. HPLC comparison of hyperforin standard (A) to hyperforin extracted from Hypericum prolificum (B) and quercetin standard (C) to quercetin extracted from Hypericum microsepalum (D). Results