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ACES Current Research:

 

Chromatographic Quantification of Medicinal Compounds in Flowering and Nonflowering Wild-Harvested Actaea racemosa L.

Amanda Vickers, Sunshine Brosi, James Howell, David Puthoff

Black cohosh (Actaea racemosa) is a perennial herb native to deciduous woodlands in Eastern North America producing a flowering raceme persistent from midsummer into the fall. Traditionally, black cohosh has been used as a specific remedy for musculoskeletal pain and to support uterine function. Today, extracts of black cohosh are marketed primarily for use in relieving hot flashes associated with menopause. A majority of the raw material which supplies the global market for black cohosh products is harvested from naturally occurring wild populations in Appalachian woodlands.  Although floral characteristics greatly assist in the ability of a harvester to differentiate between A. racemosa and the concurrent species A. pachypoda Ell. and A. podocarpa DC., there is currently no incentive for wild harvesters to select only flowering individuals. This research addresses whether harvest based on the presence of a raceme could allow a wild harvester to increase the yield of medicinal constituents in the rhizomes, while insuring correct identification. In August 2011, 40 plants were harvested, dried and weighed. Plants which had produced a flowering raceme failed to differ in rhizome mass from those which had not (p = 0.429). Rhizomes of flowering and non-flowering black cohosh plants were quantified using HPLC with PDA and UPLC with MS.  The levels of several active ingredients including actein, 23-epi-26 deoxyactein, caffeic acid, ferulic acid, and kaempferol were quantified.  In addition to yielding comparisons that are directly applicable to wild harvesting, quantitative analysis will allow comparison with both cultivated and cell-cultured plant material. Improvements in the ability to produce these compounds in vitro will also enable conservation efforts by reducing the demand on wild populations. 

Appalachian Dulcimers Project

Mitra Karimian and Sunshine Brosi, Department of Biology, Frostburg State University

The sounds of Appalachian dulcimers rings with a distinctive and charming sound that no other instruments can produce.  As a folk instrument it has been prevalent in both Northern and Southern Appalachia for centuries from Germanic and Scotch-Irish traditions respectively.   Production and form of the Appalachian dulcimer is highly variable, as is the sound and style of play(Ritchie 1964).  Lutiers, or stringed instrument artisans, primarily rely on locally-abundant species for production.  Ethnographic research has answered how materials for this folk art are shifting due to changes in Appalachian forests.  Students from Frostburg State University completed interviews and questionnaires on materials for construction (IRB # FSU-H2010-011).  Prominent species selected included black walnut, black cherry, sassafras, yellow-poplar and sugar maple.  Missing from previous accounts are American chestnut, pine, spruce, birch (Seeger 1985) and butternut (Wigginton 1975).  Many of these species are being lost from our forest due to exotic pests.  Three-quarters of zithers collected wood themselves locally which is consistent with reports from 35 years prior (Wigginton 1975) and most who purchased wood spent an average of $25 per wood for each dulcimer.  Dulcimers ranged in sale price from $180 to $700 and half also sold furniture. Zithers are able to adjust materials for dulcimers due to forest dynamics.  However, as additional species become less abundant, or local small-woodlots more scarce, many may be required to purchase wood.  Purchasing wood for dulcimer making may make the process less economically viable and reduce the cultural knowledge surrounding tonal quality and species characteristics.

 

Increasing Active Compounds in Black Cohosh

David Puthoff, Frostburg State University

 Plants produce many secondary metabolites and most of these are targeted to defending the plant from insect or microbe attack. These secondary metabolites have been used by humans for many years and purposes including treatments for ailments and ceremonial purposes. Black cohosh is a well known traditional medicine for the treatment of several ailments (Tyler, 1993).  Most of the black cohosh product comes from wild populations with commercial production rarely occurring (Persons and Davis, 2005). Over harvesting of wild black cohosh populations could lead to reduced genetic diversity, elimination of small populations and destruction of forest ecosystems from the disturbances caused by digging up the plants. One method that could be useful for decreasing the number of plants needed to supply the current market demand would be to identify ways to increase the active ingredient(s) levels within harvested rhizomes. This can be accomplished several ways. One way would be to simulate attack by insects; thus causing the plant to produce more secondary compounds, such as actein and deoxyactein.  Currently, experiments are under way to elucidate whether active ingredient (actein and deoxyactein) levels after simulated insect attack increase, decrease or stay at pre-wound levels. These experiments will simulate an insect attack by crushing the rhizome. After 2 or 4 days, the rhizomes will be dried and active compounds extracted. The active compounds will be quantifiedusing HPTLC (high performance thin layer chromatography). If these methods are successful in increasing active ingredients, harvesters may need to dig fewer plants. This will help conserve populations and genetic diversity.

Black Cohosh Habitat Modeling

Sunshine Brosi, Frostburg State University, and Jim Howell, Allegany College of Maryland

Black cohosh, Actaea racemosa L., is a perennial understory herb found in Appalachia. Current increasing demand is as a natural alternative to menopausal hormone therapy. Because this plant primarily wild harvested, concerns have been raised regarding overharvest and harvest sustainability. The Brosi lab is focused on determining factors related to sustainability and restoration of rare, threatened, and endangered species. Current study objectives are to increase the understanding of black cohosh habitat requirements, including guiding forest management plans for

non-timber forest products (NTFPs). Objectives also include evaluation of the plant’s ecological communities, current availability, health, and reproduction of the resource. Known populations of black cohosh in western Maryland were analyzed for multiple environmental factors. From known occurrences, Mahalanobis distance techniques were used to predict the location of additional populations. Conditions most likely to predict populations were determined using GIS data layers. 115 populations were found in Potomac-Garrett State (PGSF), Savage River (SRSF) and in Green Ridge (GRSF) State Forests in western Maryland. 73% of the observed locations were classified as ideal habitat. Model testing using bootstrapping resulted in a predication accuracy of 82%. Ideal habitat occurred on lower slopes, higher values of topographic convergence index, smaller “distance to stream” values and higher pH than average. suitable habitat constitutes up to 30% of SRSF and PGSF, while GRSF has less than 10% suitable. Current modeling is incorporating stand age information to assist in providing baseline data for use in broader management plans that take into account economically valuable NTFPs. Habitat variables significant in predicting black cohosh locations could also be expanded to related rare and endangered species, including mountain bugbane and Appalachian bugbane. This technique is appropriate for multiple medicinal crops and has been used to model bloodroot and American ginseng. Brosi’s research also focuses on methods of cultivation to reduce demand for wild harvesting populations and to increase economic options for rural Appalachians. Over 128,000 seeds were collected from individual open-pollinated black cohosh plants in different physiographic regions of western Maryland.  Bleach treatments showed increased germination when compared to controls.  Investigations are currently determining shade requirements and companion plants. 

Biotechnology Research on Appalachian Medicinal Plants

The University System of Maryland’s Institute of Bioscience and Biotechnology Research (IBBR) (formerly the University of Maryland Biotechnology Institute’s (UMBI) Center for Advanced Research in Biotechnology (CARB)). Ed Eisenstein, Martin Spiering, and Bhavneet Kaur

Research in ethnobotany at UM is focused on identifying the genes and enzymes that are involved in the biosynthesis of key natural products produced by a number of important medicinal plants of Appalachia.  The goal of our efforts is to understand the interplay of genetic and environmental factors that are involved in the production of medicinal metabolites, and to apply that knowledge to improve the phytochemical quality of plants.

Our initial efforts have been directed towards Actaea racemosa (L. Nutt.), or black cohosh.  The roots and rhizomes of black cohosh are harvested for commercial medicinal purposes because they contain bioactive secondary metabolites or natural products.  Although there is a rich ethnobotanical tradition for the use of black cohosh for a variety of conditions, more recently it has become popular as a treatment for vasomotor menopausal symptoms, especially since the Women’s Health Initiative described such adverse effects as increased risk of breast cancer and cardiovascular disease from hormone replacement therapy.  Additionally, black cohosh extracts are being examined for their effects on breast and prostate cancer models.  However, the effect of black cohosh on menopause or cancer based on a variety of clinical trials, its safety, and its mechanism of action, remain unclear and is under intense investigation.

Our approach has focused on developing a tissue culture system to generate a useful laboratory model for black cohosh, establishing cDNA libraries for a variety of tissues in the plant, using transcriptional profiling and LC/MS approaches to develop gene-metabolite maps for the production of cimiracemates, terpene glycosides and tryptoamine derivatives in planta, as well as protein production for structural and functional analysis of the enzymes involved in metabolite biosynthesis. This year we discovered the critical conditions needed to maintain black cohosh tissue culture cells in the laboratory.  This breakthrough, developed for the first time for any species in the Ranunculaceae family, will enable us to more easily identify the signals needed to elicit the production of different classes of metabolites, and in that way allow us to more rapidly probe gene-enzyme-metabolite relationships.

We are also examining a number of other important medicinal plants that are attractive targets for wildcrafting and agroforestry in the Appalachian region.  In addition to ginseng and black cohosh, among the most important medically and with the most economic potential are bloodroot (Sanguinaria canadensis), goldenseal (Hyrastis canadensis), false unicorn (Chamaelirium luteum) and blue cohosh (Caulophyllum thalictroides).  Our general plan is to collect germplasm and certified plant specimens for bloodroot, goldenseal, false unicorn and blue cohosh in an effort to maintain a supply of plants on site, and to develop culture systems to enable the expansion of our efforts in developing gene-metabolite networks in black cohosh to other important Appalachian plants. 

 

 The Impact of Artificial Shade Cloth and Substrate on the Establishment Success of Cultivated Actaea racemosa L. in Western Maryland, USA

Robbie Kutchman, Sunshine Brosi, Ph.D, James Howell, Ph.D

Frostburg State University

 Actaea racemosaL. (black cohosh) has been popularized relatively as an herbal treatment for symptoms associated with menopause. Et situ conservation efforts and commercial cultivation are uncommon with this species to date.  As demand increases it is essential to determine optimal conditions for survival, growth, and production of active constituents.  However, limited information exists on effective methods of cultivation of black cohosh (Fischer et al. 2006). Previous studies have shown increasing actein and deoxyactein concentrations under increasing light using 78% artificial shade and forested areas (McCoy et al. 2007).  Questions remain of the ideal shade cloth percentage for production in non-forested areas.  Rhizomes from two sources were transplanted under 50, 60, 70, and 80% artificial shade. The mortality rate determined in August 2010 under shade conditions is 35.3%. The full sun plots at both sites suffered a higher mortality of 67% due to drought conditions. This contrasts a previous study under irrigated conditions which correlated successful growth in full sun (Persons and Davis 2005).

One of the main factors affecting shoot presence for this study was initial rhizome weight. Rhizomes with an initial weight of less than 10g experienced 90% mortality, while rhizomes weighing above 50g had over 50% shoot presence.  Initial rhizome size was significantly correlated with plant height (r2= 0.13, p=0.0446), but not diameter (r2=.073, p=.2653). Between artificial shade percentages there were no significant differences in survival height or diameter (p >.05).  Future studies include evaluating the material for active constituents and soil analysis at both sites.  This research will aid in identifying factors for et situ conservation and cultivation for this important medicinal herb.  

Pollinators of Black Cohosh and Network Analysis

Dan Fiscus, Frostburg State University Department of Biology

Identification of species, their interactions, and the relative magnitude of those interactions, provides data for ecological network analysis of trophic, pollination and other relationships. Characterization of the insect pollinators and herbivores associated with black cohosh (Actaea racemosa) is providing greater understanding of its ecological relationships and to characterize its ecological niche. This includes identification of species on which black cohosh is dependent for reproduction and propagation (for example, several species of bumblebees), and those species that are dependent on black cohosh. These species include the Appalachian azure butterfly (Celastrina neglectamajor), whose caterpillars feed only on black cohosh. While the specific pollinators are known for related plants in the genus Actaea, they are not known for black cohosh. With Dr. Kristen Lennon and her student, pollen loads from bees captured on black cohosh flowers were examined under scanning electron microscope (SEM) and much of the pollen confirmed to be from black cohosh. In field work completed in the summer of 2010, 20 species of bees and flies were captured and identified via collaboration with Sam Droege at the Patuxent Wildllife Research Center. These include two native species of Bombus, the bumblebees (Bombus impatiens and Bombus bimaculatus), seven native species from the genus Lasioglossum (small black sweat bees, including Lasioglossum heterognathum), non-native honeybees (Apis mellifera) and other bee species in the genera Hylaeus and Augochlora as well as Syrphid flies.

 

Data on visitation rates and eventual fruit development will be analyzed to correlate frequency of visits by bee species and seed production. Pollen will also be identified on the captured bees with assistance of Dr. Phil Allen in the FSU Geography Department. This information will help determine the functional importance of each species in the pollination of black cohosh. Additional results from 2010 include creation of many digital videos of visits and behavior of insects on black cohosh flowers. Many of these videos are available online at http://www.youtube.com/user/dafiscus. In addition to continued efforts to identify pollinators and their pollen loads, in 2011 insect exclusion bags will be placed on flowers at multiple sites. Examination of flowers and/or fruits afterward will tell the degree to which black cohosh is dependent on insects for pollination, and reveal any self-pollination capacity. This work has been aided by collaboration of Maryland State Botanist, Chris Frye.

 

Understanding pollination ecology is essential for black cohosh, and comprehensive knowledge of the ecological web of relationships is equally essential for any species of medicinal plant. Knowledge of essential ecological relationships of black cohosh and many of the key mutualistic insect species with which it has co-evolved and currently co-exists will make it possible to propagate, manage and conserve black cohosh in a holistic and sustainable fashion. While this research has focused on networks of pollination relationships, network analysis can also be used to study other ecological networks in forest ecosystems (e.g., carbon and nitrogen fluxes) and can also aid understanding of 1) human roles in regional ecosystems and 2) systemic structure and function in social and economic networks.  This research has benefited from ACES funding and from the assistance of many ACES faculty, staff and students, private landowners and others.

 

Evaluation of solvent affects on anti-oxidant capacity levels of Black Cohosh

Student Marcus Carter, Assistant Professor Peggy S Biser PhD. Department of Chemistry, Frostburg State University, Frostburg, Maryland, United States

Black cohosh is a traditional medicinal herb that has been used to alleviate menopausal symptoms. As studies continue, new uses of the herb have been proposed such as a preventative of bone-loss and an anti-inflammatory. The chemical properties and proposed mechanisms that provide the meditative effects of cohosh may be linked to the compound's high level of anti-oxidants. The study will investigate the influence of various reconstitution solvents on the total anti-oxidant levels determined by the ORAC- fluorescien (ORAC-FL) assay.