2003 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? Natural products offer a vast and virtually unlimited source of new agents for both the pharmaceutical and agrochemical industries. The National Center for Natural Products Research (NCNPR) was created to bring together an alliance of academia, government, and the pharmaceutical and agrochemical industries to integrate research, development, and commercialization of potentially useful natural products. The overall goal of this project is to discover new bioactive natural products from higher plants with potential to become lead compounds for development as new agrochemicals and pharmaceuticals, and to examine the biological and chemical properties of medicinal plants in order to develop them as alternative crops. The specific research objectives include: 1. Develop strategies for rational collection of higher plants as a source of novel bioactive natural products 2. Isolate and identify novel anticancer, antiinfective, and antiinflammatory/immunomodulatory natural products from higher plants 3. Develop methods to authenticate, pharmacologically characterize, and chemically analyze botanical dietary supplements and medicines from plants 4. Investigate agronomics, cultivation, harvesting, and processing of medicinal plants 2.How serious is the problem? Why does it matter? Despite significant advances in drug discovery in the last half-century, there remain important unmet therapeutic needs. Likewise, there is an urgent need to identify new and safer pest management agents. Natural products offer a virtually unlimited source of potential new pharmaceuticals and agrochemicals, largely because of the remarkable diversity of both chemical structures and biological activities of naturally occurring secondary metabolites. Over 50% of prescription drugs on the US market were derived from natural products. Additionally, the utility of novel bioactive natural products as biochemical probes, the development of novel and sensitive techniques to detect biologically active natural products, improved techniques to isolate, purify, and structurally characterize these active constituents, and advances in solving the demand for supply of complex natural products collectively provide a compelling justification to search for bioactive natural products with potential pharmaceutical and agricultural applications. Ultimately, commercially available plant-derived pharmaceuticals and agrochemicals could create a demand for such plants as alternative crops. Additionally, a wide array of plant-based substances (i.e., botanical dietary supplements, 'herbal remedies') are consumed by millions of people daily in the US. The US market for botanical dietary supplements is estimated to be between 4 and $5 billion. There is a critical need for quality, research-based scientific information to provide the basis for establishing standards and monitoring the quality and safety of botanical products consumed by the public, to guide health care professionals and the consuming public in their decisions regarding the use of these products, for introducing new products that have a firm research underpinning, and for assessing the potential of such products to become alternative crops for US farmers. Currently, this information is not available and there is an urgent need for research to be undertaken to address this shortcoming. 3.How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research allows scientists from USDA and the University to work together to identify new molecular targets for the discovery of agrochemicals and pharmaceuticals and to share resources, including specimens of plants and marine organisms collected from worldwide sources, that facilitate the discovery of novel bioactive natural products that can serve as lead compounds for development of new toxicologically safe, environmentally benign pest management agents and new pharmaceuticals. This research also allows scientists from USDA and the University to work together to solve problems related to the production of high quality medicinal plants that produce drugs or precursors to drugs, or are sold as dietary supplements. 4.What were the most significant accomplishments this past year? Two patent applications were submitted with inventors from the USDA and the University. These two research projects, a new algaecide and a new class of antifungals for plants, demonstrate the unique synergisms that exist in the natural products program. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Over forty joint projects between USDA and University scientists have been initiated. These projects have led to one issued U.S. patent and 2 patent applications. In addition, 3 joint publications have originated from the collaboration through 2003. The plant specimen acquisition project has continued to expand with a total of over 15,000 samples in the natural product repository. These samples are being characterized on an ongoing basis for biological activity and the chemical constituents responsible for the biological effects are being identified and isolated. In addition, collaborations with external collection groups have been established to enhance our internal capabilities, most notably with the Missouri Botanical Garden, the New York Botanical Garden and the Kunming Institute in China. Additional biological assays, including functional genomic-based assays, have been utilized to identify potentially useful bioactive natural products. Several new biological assays for plant and human pathogens have been added. Emphasis has been placed on the discovery of antifungals, antibacterials, antiinflammatory agents and immunomodulating agents. Over 6800 natural product specimens were screened in 2002-2003 for biological activities against specific molecular targets and/or whole cell systems. Significant progress continues to be made in determining antifungal lead compounds for pharmaceutical use from several novel classes of compounds. In addition, promising agrochemical lead compounds have been identified from the sampangine class of antifungals and a joint USDA/University of Mississippi patent application has been filed. Progress continues to be made on the development of a promising 8-aminoquinolone derivative for the treatment of PCP infections in AIDS patients and malaria. Scale-up of the compound is ongoing in collaboration with the National Institutes of Health (NIH). We continue to develop new analytical and biological screening methods to characterize the chemical constituents responsible for biological activity of botanicals consumed as dietary supplements. This work forms the foundation for all studies that determine the correlation of pharmaceutical profiles and chemical profiles with observed therapeutic effects and conditions of cultivation and harvesting of medicinal plants. Work continues on a grant obtained from the NIH to continue our research on Echinacea and collaboration with USDA to determine genetic variability in several medicinal plants of commercial interest. This project is aimed at understanding genetic and phenotypic factors that contribute to consistent high quality medicinal plant material, including the optimal conditions for the cultivation, harvest, and post-harvest processing and storage of medicinal plants to ensure maximum yield of valuable chemicals and/or therapeutic effect. Two license agreements were executed with pharmaceutical companies to commercialize potent immunostimulants isolated from blue green algae and aloe. Field trials are ongoing in collaboration with Mississippi State University on high-yielding Mayapple cultivars. Demonstration plots of several other medicinal herbs are being grown in our Medicinal Plant Garden. Pond trials were initiated on the anthraquinone derivative as a potential algaecides. A jointly owned USDA/University of Mississippi patent application was filed on the compound that has been shown to have selective activity against the blue green algae responsible for the development of the off-flavor in catfish. Additional pond trials will be conducted this summer. Discussions are ongoing with a potential licensee for the use of this compound. The collaborative research project with the Center for Food Safety and Applied Nutrition at the FDA was expanded to include biological testing. Several plants of interest including Ephedra species have been collected and characterized. The University planned and hosted a workshop on authentication of botanicals as part of the FDA collaboration in August 2002. The University is planning a workshop for September 2003 on hepatotoxicity assessment of botanicals. 6.What do you expect to accomplish, year by year, over the next 3 years? During the next year, we will continue our studies related to each of the four major objectives of this project: developing strategies for collection of plants yielding bioactive natural products, isolating and identifying novel bioactive natural products, understanding the biological and chemical properties of medicinal plants, and developing medicinal plants as alternative crops. Specifically, we plan to work with our collaborators at other institutions to collect plant material from geographic areas and taxa not currently represented in our existing repository of samples. We will evaluate more than 2000 natural product samples for biological activities against specific molecular targets and/or whole cell systems. We will also continue to develop new antifungal lead compounds as well as progress the aminoquinolone derivative for through preclinical studies. We will continue collaboration with USDA on over 20 projects. We will work continue to work with the FDA on the characterization of high priority plants. We will transfer analytical methods to FDA. We will host a workshop on hepatotoxicity assessment of botanicals in September 2003. We will continue field studies on Mayapple cultivars and several other popular medicinal plants. Commercialization efforts will be continued to find a partner for the podophyllotoxin process technology and to finalize licensing agreements on the algaecide, and taxol technologies. 7.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Procedures for isolating purified reference compounds from popular dietary supplement ingredients and analytical methods for characterizing the compounds have been licensed to ChromaDex, Incorporated. ChromaDex is currently selling these standards to the dietary supplement and functional food industries. A license agreement has been in the final stages of review for over 1 year with an industrial partner in Canada for a process for extracting taxanes from various taxol species. Licensing is pending the issuance of a patent in Canada. We are currently addressing issues raised by the Canadian patent office. It is anticipated that the taxol technology will be transferred to the industrial partner for use in production of taxanes during the fall of 2003 if the patent is granted. A process for extracting a novel immunostimulatory ingredient from blue green algae was transferred to Nordic Phytopharma. It is anticipated that a product based on the blue green algae technology will be commercially available in 2004 assuming successful completion of the development studies. A process for extracting a novel immunostimulatory ingredient from aloe was transferred to AHC, Inc. It is anticipated that a product based on the aloe technology will be commercially available in 2004 assuming successful completion of the development studies. The results of our research are disseminated through presentations at scientific meetings, publications in scientific journals, and/or patents. The information is thus available to the scientific community immediately upon presentation or publication. For patented technology, efforts to identify licensing partners commence immediately upon filing for patent application (or sooner in some cases), and it is expected that it will be 5 - 10 years before the technology (especially new pharmaceuticals) will be available to the end user. For work related to the development of processes for recovery of useful plant constituents from medicinal plants (e.g., Mayapple project), the availability to the end user could be much sooner, probably 2 - 3 years. Constraints in developing pharmaceuticals are related principally to the rigorous regulatory requirements mandated by the Food and Drug Administration. The cost of developing a pharmaceutical product has been estimated to exceed $800 million. In order for a pharmaceutical company to be interested in licensing one of our lead compounds we must conduct sufficient pharmacology and toxicology studies to give an indication that the compound is a likely candidate for full development and has advantages over existing drugs and compounds in industrial pipelines. 8.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Al-Rehaily, A.J., M.S. Ahmad, et al. (2002). "New Axane and Oppositane Sesquiterpenes from Teclea nobilis." Journal of Natural Products 65(9): 1374-1376. Bedir, E., H. Lata, et al. (2003). "Micropropogation of Hydrastis canadensis: Goldenseal, a North American Endangered Species." Planta Medica 69: 86-88. Chambliss, W.G., C.D. Hufford, et al. (2002). "Assessment of the Quality of Reference Books on Botanical Dietary Supplements." Journal of the American Pharmaceutical Association 42(5): 723-734. Dayan, F.E., J.M. Kuhajek, et al. (2003). "Podophyllum peltatum Possesses a ß- Glucosidase with High Substrate Specificity for the Aryltetralin Lignan Podophyllotoxin." Biochimica et Biophysica Acta 1646: 157-163. Duncan, C.J.G., M. Cuendet, et al. (2003). "Chemical and Biological Investigation of the Fungus Pulveroboletus ravenelii." Journal of Natural Products 66(1): 103-107. Duncan, C.J.G., N. Pugh, et al. (2002). "Isolation of a Glactomannan that Enhances Macrophage Activation from the Edible Fungus Morchella esculenta." Journal of Agricultural and Food Chemistry 50: 5683-5685. El Sayed, K., M.Yousaf, et al. (2002). "Microbial and Chemical Transformation Studies of the Bioactive Marine Sesquiterpenes (s)-(+) Curcuphenol and -Curcudiol Isolated from a Deep Reef Collection of the Jamaican Sponge Didisdcus oxeata." Journal of Natural Products 65(11): 1547-1553. Feltenstein, M.W., L.C. Lambdin, et al. (2003). "Anxiolytic Properties of Piper methysticum Extract Samples and Fractions in the Chick Social-Separation-Stress Procedure." Phytotherapy Research 17: 210-216. Ferreira, D. and X.C. Li. (2002). "Oligomeric Proanthocyanidins: Naturally Occurring O-Heterocycles." Natural Product Reports 19: 517-541. Ganzera, M., M.I. Choudhary, et al. (2003). "Quantitative HPLC Analysis of Withanolides in Withania somnifera." Fitoterapia 74: 68-76. Ganzera, M., J. Zhao, et al. (2002). "Chemical Profiling and Standardization of Lepidium meyenii (Maca) by Reversed Phase High Performance Liquid Chromatography." Chem. Pharm. Bull. 50(7): 988-991. Gochfield, D., E. El Sayed, et al. (2003). "Marine Natural Products as Lead Anti-HIV Agents." Mini Reviews in Medicinal Chemistry 2003(3): 451-475. Herath, W.H.M.W., D. Ferreira, et al. (2003). "Microbial Transformation of Xanthohumol." Phytochemistry 62: 673-677. Hossain, C.F., M.R. Jacob, et al. (2002). "Gentipatriol, a New Cycloartane Triterpene from Genipa Spruceana." Journal of Natural Products 66 (3): 398-400. Jardat, M.S., D.J. Noonan, et al. (2002). "Pseudolaric Acid Analogs as a New Class of Peroxisome Proliferator-Activated Receptor Agonists." Planta Medica 68: 667-671. Kumar, D., M.R. Jacob, et al. (2002). "Synthesis and Evaluation of Anticancer Benzoxazoles and Benzimidazoles Related to UK-1." Bioorganic and Medicinal Chemistry 10: 3997-4004. Lalchandani, S.G., L. Lei, et al. (2002). "Yohimbine Dimers Exhibiting Selectivity for the Human Alpha-2C-Adrenoceptor Subtype." Journal of Pharmacology and Experimental Therapeutics 303(3): 979-984. Li, X.C. and D. Ferreira (2002). "Stereoselective Cyclization of Stilbene Derived Carbocations." Tetrahedron 59: 1501-1507. Li, X.C., A.S. Joshi, et al. (2002). "Absolute Configuration, Conformation, and Chiral Properties of Flavanone-(3?8")-Flavone Biflavonoids from Rheedia acuminata".Tetrahedron 58: 8709-8717. Li, X.C., A.S. Joshi, et al. (2002). "Fatty Acid Synthase Inhibitors from Plants: Isolation, Structure Elucidation, and SAR Studies." Journal of Natural Products 65(12): 1909-1914. Luo, Y., J.V. Smith, et al. (2002). "Inhibition of Amyloid-ß Aggregation and Caspase-3-Activation by the Ginkgo biloba Extract EGb761." PNAS 99(19): 12197-12202. Mineno, T., K.M. Stanford, et al. (2002). "Solution-Phase Parallel Synthesis of an Isoflavone Library for the Discovery of Novel Antigiardial Agents." Combinational Chemistry & High Throughput Screening 2002(5): 481-487. Muhammad, I., S. Takamatsu, et al. (2003). "Cytotoxic Sesquiterpene Lactones from Centaurothamnus maximus and Vicoa pentanema." Phytotherapy Research 17: 168-173. Nagle, D.G. and Inderjit. (2002). "The Chemistry and Chemical Ecology of Biologically Active Cyanobacterial Metabolites." Chemical Ecology of Plants: Allelopathy in Aquatic and Terrestrial Ecosystems. Inderjit and Mallik, A.U., Birkhauser Verlag. 33-56. Nagle, D.G., M.S. Slattery, et al. (2003). "Chemical Ecology and Natural Products from Wetlands: Emerging Perspectives." Achieving Sustainable Freshwater Systems: A Web of Connections. Holland, M.M., E. Blood, et al., Editors, Island Press: 157-169. Peng, J., K. Walsh, et al. (2002). "The New Bioactive Diterpenes Cyanthiwigins E-AA from the Jamaican Sponge Myrmekioderma styx." Tetrahedron 58: 7809-7819. Puglisi, M.P., V.J. Paul, et al. (2002). "Co-occurrence of Chemical and Structural Defenses in the Gorgonian Corals of Guam." Marine Ecology Progress Series 239: 105-114. Rogers, P.D., K.S. Barker, et al. (2003). "Heat-Induced Superaggregation of Amphotericin B Attenuates its Ability to Induce Cytokine and Chemokine Production in the Human Monocytic Cell Line THP-1." Journal of Antimicrobial Chemotherapy 51: 405-408. Schaneberg, B.T., S. Crockett, et al. (2003). "The Role of Chemical Fingerprinting Application to Ephedra." Phytochemistry 62: 911-918. Takamatsu, S., T.W. Hodges, et al. (2003). "Marine Natural Products as Novel Antioxidant Prototypes." Journal of Natural Products 66(5): 605-608. Xie, D.Y., S.B. Sharma, et al. (2003). "Role of Anthocyanidin Reductase, Encoded by BANYULS in Plant Flavonoid Biosynthesis." Science 299(17 January 2003): 396-399. Yousaf, M., K.A. El Sayed, et al. (2002). "12,34 Oxamanzamines, Novel Biocatalytic and Natural Products from Manzamine Producing Indo-Pacific Sponges." Tetrahedron 58: 7397-7402. Zhang, Z., H.N. ElSohly, et al. (2003). "Phenolic Compounds from Nymphaea odorata." Journal of Natural Products 66(4): 548-550. Zhang, Z., H.N. ElSohly, et al. (2002). "Flavanone Glycosides from Miconia traili." Journal of Natural Products 66(1): 39-41.