Source: FROSTBURG STATE UNIVERSITY submitted to
APPALACHIAN CENTER FOR ETHNOBOTANICAL STUDIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0214913
Grant No.
2008-38922-19357
Project No.
MDW-2008-03462
Proposal No.
2008-03462
Multistate No.
(N/A)
Program Code
AA-X
Project Start Date
Aug 1, 2008
Project End Date
Jul 31, 2010
Grant Year
2008
Project Director
Hoffman, J.
Recipient Organization
FROSTBURG STATE UNIVERSITY
(N/A)
FROSTBURG,MD 21532
Performing Department
(N/A)
Non Technical Summary
Frostburg State University, West Virginia University, and the University of Maryland Biotechnology Institute have partnered to create the Appalachian Center for Ethnobotanical Studies (ACES). This is a collaborative, inter-institutional effort devoted to the multidisciplinary study and conservation of native plants. A primary focus of ACES is to foster economic growth in the region through the managed development of the area's natural resources. It will work with existing businesses and facilitate the development of new local enterprises to explore the use of regional plants for health-related purposes. It will also help to document and preserve Appalachian culture as it relates to wild plant harvesting and herbal medicine through community outreach and education programs. Specific objectives are: Scientific Research Identify and analyze native plants having medicinal and cultural significance by integrating classical growth and harvesting of ethnobotanical plants with herbal practice and with modern biotechnology methods. Investigate the germination and propagation characteristics of native plants having medicinal and cultural significance to develop standards for the industry. Apply laboratory research findings to conserve, propagate, validate, and market native Appalachian plants for medicinal and other purposes. Involve university students in ethnobotanical research both at under and graduate levels. Economic Development and Public Outreach Develop partnerships between universities, local growers, and harvesters to enhance the economic development of the region's ethnobotanical industry. Encourage conservation by working with local communities to manage commercial sustainable private cultivation and harvesting of native plants having medicinal use and applications. Foster development of new enterprises that focus on the region's ethnobotanical industry. Educational Outreach Preserve Appalachian culture by documenting and developing education curricula on traditional plant harvesting practices and folk medicine. Develop K-12 curricula for Appalachian schools to achieve mandated learning objectives using regionally familiar materials and scenarios. Develop online, web based education modules for public outreach and distance learning. The proposed project will enable ACES to hire a Research Director to coordinate ongoing research efforts of the three partner institutions, while also focusing on establishing relationships with growers and harvesters in Appalachia. Post-doctoral fellows at both UMBI and FSU will further the research on the potential medicinal properties of black cohosh already begun at these institutions. ACES will also expand its economic development outreach efforts and educational offerings, including additional workshops and symposia for growers and harvesters.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1232220100015%
1232220106010%
1232235100015%
1232235106010%
6082220100015%
6082220106010%
6082235100015%
6082235106010%
Knowledge Area
123 - Management and Sustainability of Forest Resources; 608 - Community Resource Planning and Development;

Subject Of Investigation
2220 - Medicinal crops, non-narcotic; 2235 - Herbs and spices;

Field Of Science
1060 - Biology (whole systems); 1000 - Biochemistry and biophysics;
Goals / Objectives
Identify Appalachian plants with potential medicinal or other useful properties with the aim of discovering novel molecules that have therapeutic or other applications. Encourage natural resource conservation by working with local families and communities to manage commercial harvesting of wild plants, cultivate economically important plants whenever possible, and create an awareness of the critical need to insure sustainability by avoiding extractive harvesting practices. Preserve Appalachian culture by exploring the relationship of individuals, families, and communities to the harvesting of wild plants and documenting their traditional use of botanicals. Integrate the indigenous knowledge of Central Appalachians with modern molecular tools and technologies developed at UMBI's Center for Advanced Research in Biotechnology, plant ecology and physiology studies at Frostburg State University and the West Virginia University Center for Wildlife Habitat and Medical School's Department of Alternative Medicine. Foster economic development by collaborating with pharmaceutical companies to explore the potential for developing compounds derived from Appalachian plants for therapeutic or nutraceutical purposes, emphasizing equitable financial return to the region. Involve university undergraduate and graduate students in program related research in biology, chemistry, environmental sciences, sociology, and anthropology. Develop K-12 science curricula for Appalachian schools that teach scientific principles and concepts and achieve mandated learning objectives through use of regionally familiar materials and scenarios. Develop online, web based education modules for public outreach and distance learning.
Project Methods
1. Identify, sample and archive germplasm from populations of Black Cohosh in Western Maryland. Site coordinates will be documented using GPS technology. Germplasm will be sent to the Agricultural Research Service. Rhizmome samples will be obtained for chemical and functional analysis. 2. Chemically fingerprint rhizome samples obtained locally and throughout the Appalachian chain, using Soxlet extraction with three different solvent systems. The systems are 75% ethanol, 40% isopropanol and 100% methanol. These extracts are concentrated and then subjected to LCMS. We will obtain fingerprints of two classes of chemicals that have been identified as potentially important: triterpenes and phenolics. We also seek to quantify specific chemicals that have shown promise: cimifugoside, actein, 27-deoxyactein, caffeic and ferulic acids. 3. Functional studies of Black Cohosh rhizome extracts. Using the human promyelocytic leukemia cell line, we will look at the potential impact of the content of the various alcoholic extracts on the differentiation of these cells. 4. Develop a habitat model based on existing locations of Black Cohosh. This will include soil nutrient levels, with a particular emphasis on calcium. Factors such as geology, light and shade, precipitation, temperature and soils will be considered using both on site and satellite imagery. 5. Ecological network Analysis of Black Cohosh. We will identify the species that eat or parasitize Black Cohosh, then estimate the energy, carbon and/or nitrogen transferred to each of the species via herbivory or parasitism. Standing stocks for Black Cohosh and each linked species, as well as abiotic stocisks providing inputs to Black Cohosh will be estimated. Available software will be used to calculate important community-level and network indices on the forest ecosystems in which Black Cohosh lives. 6. Generate a tissue culture system for black cohosh. We will establish stable cell cultures of black cohosh using Agrobacterium rhizogenes to generate hairy root cultures. 7. Utilize UPLC methods and mass spectrometry analysis to screen cultures for small molecule production by treatment with signaling elicitors. 8. Construct a cDNA library from a tissue culture line that exhibits optimum production of signature compounds. 9. Undertake sequencing analysis of the EST library. 10. Design working hypotheses for the production of the small molecule metabolites of black cohosh.

Progress 08/01/08 to 07/31/10

Outputs
OUTPUTS: Eighty-three black cohosh population sites were identified in 3 state forest systems of western Maryland: Green Ridge, Savage River and Potomac. Fifty-four accessions were collected for chemical analysis, with representatives from each zone. Associated community structure has been documented on these 83 sites, including 747 herbaceous vegetation plots, 249 sapling plots, 83 overstory plots, and 498 soil samples. A habitat model based on existing locations of black cohosh has been developed. This has been used to predict habitat suitability across Garrett and Allegany counties using the Mahalanobis Distance model and GIS data. Chemical analysis has been conducted on 41 root samples from sites in Virginia and 18 root samples from sites in Maryland. A low temperature method of extraction was used, with the samples cleaned, vacuum dried, pulverized and stored. Researchers examined the effect of various solvents on the extraction of phenolic compounds from black cohosh using LC-PDA. Functional studies of black cohosh rhizome extracts were also conducted. Using the human promyelocytic leukemia cell line HL-60, researchers worked to test the ability of black cohosh extracts to induce cell differentiation. A study on the ecological relations of black cohosh was also conducted. Field data was collected on insect visitors and pollinators of black cohosh, with 80 insects captured, preserved and identified for further study. Additional outputs achieved by our collaborating partner, University of Maryland Biotechnology Institute, include: 1. An in vitro culture system for black cohosh was successfully developed. 2. Molecular and functional analysis of secondary metabolite genes in black cohosh was conducted. 3. Reliable protein expression systems for black cohosh genes have been developed. 4. Black cohosh total RNA was extracted from elicited and control leaves and reverse transcribed into cDNA, which has been used for the creation of subtractive EST libraries. 5. UMBI has worked on authentification of black cohosh plants through DNA barcoding. A molecular DNA barcoding method based on PCR amplification and sequencing of rDNA and chloroplast DNA was tested on black cohosh. The PCR-amplified DNA sequences obtained from several in-house plant stocks were compared to corresponding sequences from authentic A. racemosa and found to have excellent resolving power to distinguish black cohosh from its closely related relatives. 6. Seed germination in black cohosh was obtained using two different methods, both of which resulted in seed germination. The in vitro regenerated shoots and seedlings are being used to generate Agrobacterium rhizogenesis mediated hairy root cultures. 7. UMBI has undertaken laboratory cultivation of bloodroot, goldenseal, false unicorn, and blue cohosh with the aim of ultimately developing in vitro culture systems for these plants that would be useful in biochemical and molecular genetic analyses. An additional output of this project was bringing together growers, scientists and regulators for discussion on the progress and prospects of black cohosh as a sustainable medicinal plant through a June 2009 symposium held at UMBI in Rockville. PARTICIPANTS: Dr. Joseph Hoffman, Dean of the College of Liberal Arts and Sciences at Frostburg State University, served as principal investigator on this project, overseeing the research efforts from faculty within the various disciplines at FSU. Other key personnel from FSU were Dr. Peggy Biser from the Chemistry Department who performed the chemical analysis, Dr. Sunshine Brosi from the Biology Department who worked on locating and documenting black cohosh populations in Western Maryland, Dr. Dan Fiscus and Dr. David Puthoff from the Biology Department who conducted the analysis on ecological relations of black cohosh, and Dr. Scott Fritz from the Biology Department who worked with Dr. Biser on functional studies of black cohosh rhizome extracts. Dr. Matthew Ramspott and Dr. Francis Precht from the Geography Department began work on the habitat modeling portion of the project. In addition, Dr. James Howell from Allegany College of Maryland partnered with Dr. Brosi on locating and documenting black cohosh populations. Joseph Ferrari with the University System of Maryland Center for Environmental Studies Appalachian Laboratory also served as a partner to continue the work begun by Drs. Ramspott and Precht on the habitat modeling portion of the project. Mitra Sadeghipour was hired as a post-doctoral fellow at FSU and assisted with the chemical analysis portion of the project. A subaward was made to the University of Maryland Biotechnology Institute to generate a tissue culture system for black cohosh, conduct molecular and functional analysis of secondary metabolite genes in black cohosh, and construct a cDNA library from a tissue culture line that exhibits optimum production of signature compounds, undertake sequencing analysis of the EST library, and design working hypotheses for the production of small molecule metabolites of black cohosh. UMBI faculty participating were Edward Eisenstein and Lori Urban. In addition to the faculty working on this project, 7 undergraduate students and 2 graduate students from FSU were involved in the research. TARGET AUDIENCES: Outreach activities for this project were targeted toward growers, harvesters, and herbal practitioners in the Appalachian region. Ethnobotany and science students at Frostburg State University were also a target audience, participating in experiential learning opportunities and receiving classroom instruction related to the project research. Scientists from federal agencies and other academic institutions were also targeted as workshop participants. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The outcomes of this project include: determining management protocols for existing black cohosh sites on public lands, producing suggestions for potential black cohosh cultivation in state managed areas, and continuing collaboration with state forest systems for additional native medicinal plants. In addition, this project is being used as a teaching tool for classes at Frostburg State University and Allegany College of Maryland (Introduction to Ethnobotany and Medicinal Botany classes). Considerable knowledge was gained regarding preferred methodology for extracting phenolic compounds from black cohosh. Knowledge was also gained regarding the growth, subculture, and cryopreservation of the HL-60 cell line. Added and improved skills and capacities for ecological network analysis were also acquired. New information on 13 unique bee visitors to black cohosh was identified. Video techniques were developed as a potential alternative and less invasive approach to identifying pollinators. Outcomes of UMBI's work have been the identification of genes and enzymes that are involved in the biosyntheses of the important compounds found in black cohosh, enhanced understanding of the interplay of genetic and environmental factors that are involved in the production of medicinal metabolites, and the application of that knowledge to improve the phytochemical quality of plants.

Publications

  • Sadeghipour,M., Biser, P., Brosi, S., and Howell, J. 2010. Analysis of solvent and the environmental effect on the Phenolic contents of Black Cohosh. Proc. of the 51st Annual meeting of the American Society of Pharmacognosy and the Phytochemical Society of North America, St. Petersburg, FL.
  • Brosi, S., and Walsh, N. 2010. Stratification protocols and germination rates of black cohosh (Actaea racemosa L.) populations from western Maryland. Proc. of the Society for Economic Botany Conference, Xalapa, Mexico.
  • Brosi, S. 2010. Sustainability of an Appalachian herb, black cohosh, Actaea racemosa L.: determining factors of habitat and abundance in western Maryland. Proc. of the Society for Economic Botany Conference, Xalapa, Mexico.
  • Ferrari, J., Brosi, S., and Howell, J. 2010. Habitat suitability for the medicinal plant black cohosh (Actaea racemosa, L.) in western Maryland- a baseline study for management of non-timber forest resources. Proc. of the 2010 International Association of Landscape Ecology, US-IALE Twenty-fifth Anniversary Symposium, University of Georgia, Athens, GA.
  • Brosi, S. 2010. People and Plant Communities of the Appalachian Mountains: Ethnobotany in western Maryland. Proc. of the 33rd Annual Appalachian Studies Conference, Engaging Communities, North Georgia College and State University, Dahlonega, GA.
  • Brosi, S. and Walsh, N. 2010. An Herb Grows in Appalachia: Sustainability of Black Cohosh Populations in Western Maryland. Proc. of the 33rd Annual Appalachian Studies Conference, Engaging Communities, North Georgia College and State University, Dahlonega, GA.
  • Minnick, A.J. and Biser, P. 2010. Black cohosh: Soxhlet extraction vs. low temperature method. Proc. of the 239th ACS National Meeting, San Francisco, CA.
  • Brosi, S. 2009. Healthy Women and Healthy Forests: Prospects for Sustainable Management of an Ethnobotanical Herb, Black Cohosh, in Western Maryland. Proc. of the Southeastern Womens Studies Association (SEWSA) Annual Conference, Appalachian State University, Boone, NC.
  • Brosi, S. and Walsh, N. 2009. Conserving Womens Herbal Healing Wisdom: Protecting Black Cohosh in Eastern Forests. Proc. of the SEWSA Annual Conference, Appalachian State University, Boone, NC.
  • Minnick, A.J. and Biser, P. 2009. Comparison of conventional Cimicifuga racemosa Soxhlet extraction technique to a low temperature alternative method. Proc. of the 237th ACS National Meeting, Salt Lake City, UT.