RESEARCH WEBPAGE

Nadja Cech

Assistant Professor

Department of Chemistry and Biochemistry

University of North Carolina Greensboro (UNCG)

Broad Research Goals

· Elucidate the mechanisms by which complex mixtures of plant compounds interact to modulate biological activity.

· Study the influence of metabolism on the immune-stimulating activity of medicinal plant constituents.

· Develop novel approaches for the chemical characterization of medicinal plants and plant mixtures using mass spectrometry and high performance liquid chromatography.

· Provide new insights into the fundamentals of electrospray ionization mass spectrometry for the analysis of small molecules

1. UNCG Mark Crosswhite works with Dr. Nadja Cech on a high performance liquid chromatograph/ electrospray ionization mass spectrometer.

2. UNCG undergraduate student Maqueita Eleazer prepares samples for analysis.

3. UNCG undergraduate student Brandie Craven prepares Echinacea plants for extraction.

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Links

· Significance of Research

· Mass Spectrometry for the Analysis of Biological Compounds

· Research Group

· Instrumentation

· Funding

· Publications

Significance of Research

Since the beginning of life on earth, plants have evolved a complex array of compounds that serve the purposes of protection against predators and attraction of beneficial organisms. These plant chemicals can profoundly influence the biochemical processes within other life forms, including humans. Because of this, there is a long history of the use of plants as medicine by peoples around the world. Today, the use of plant-based medicines in North America is on the rise, and research focusing on botanical medicines has become increasingly important.

Historically, drug development research has focused on the identification of a single constituent responsible for the activity of a plant that can be isolated or synthesized, then patented for the treatment of particular illnesses. This approach has led to the discovery of a wide variety of plant-derived drugs, such as digoxin (from foxglove, Digitalis purpurea), vinblastine (from to periwinkle, Vinca minor), and taxol (from Pacific yew, Taxus brevifolia). Over the past decade, though, as the popularity of alternative medicine in the US has increased, there has been more research into the efficacy of whole plant extracts as opposed to isolated constituents. Proponents of the use of herbal extracts suggest that their complex composition yields enhanced efficacy due to synergistic interactions among multiple components. However, this complexity makes research correlating chemical composition of extracts with biological activity extremely challenging. Our research addresses this problem with the use of sophisticated analytical instrumentation, which facilitates detailed characterization of the constituents of complex plant extracts. This detailed chemical characterization, combined with various in vitro assays, gives us insight into how complex mixtures of plant compounds interact within the human body. The ultimate goal of our research is to develop novel approaches for the use of plant medicines in the treatment and prevention of disease.

Mass Spectrometry for the Analysis of Biological Compounds

The most difficult part of the process of analyzing a plant is identifying the molecules of interest. A technique known as mass spectrometry can be very helpful in accomplishing this goal. With mass spectrometry, the weight of a molecule is determined. The molecule is then fragmented, and the weights of the fragments are measured as well. Each molecule will have a different molecular weight and will form different fragments. Thus, knowing the weights of a molecule and its fragments allows one to identify the molecule.

Molecules to be studied with mass spectrometry must be charged and transferred to the gas phase (a process known as ionization). The challenge in studying biological molecules like plant constituents is in this ionization process. Most biological molecules are sticky, and cannot be introduced into the gas phase by heating alone. Imagine heating a sample of blood or a plant leaf. The molecules in this sample will not evaporate, although they may eventually decompose.

Electrospray Ionization Mass Spectrometry (ESI-MS) is a relatively new technique that accomplishes ionization of non-volatile molecules. It is ideal for studying complex plant extracts since it can be used to analyze a variety of different compounds classes that they contain, including proteins, oligonucleotides, oligosaccharides, and small organic molecules. In addition, ESI-MS can be coupled directly to separation techniques like high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), facilitating the separation of these very complex mixtures.

Research Group

This research is carried out with a diverse and dynamic team of undergraduate and graduate students. Currently the group consists of Bethany Amber Hovater (undergraduate student at UNCG), Kathryn Tarney (undergraduate student at UNCG), Kevin Spelman (graduate student, University of Exeter, research associate at UNCG), Stacy Stinson (graduate student, UNCG), and Stacy Dee (graduate student, UNCG). We are also fortunate to collaborate with a number of faculty members at UNCG and elsewhere, including Dr. Cynthia Wenner (Research Assistant Professor in immunology, Bastyr University), Dr. Elizabeth Lacey (Professor of Biology, UNCG), Dr. Gregory Raner (Associate Professor of Biochemistry, UNCG), and Dr. Mufeed Basti (Assistant Professor, Physical Chemistry, North Carolina Agricultural and Technical University).

Instrumentation

· LC/ESI-MS: Ion trap mass spectrometer with electrospray ionization source (LCQ Advantage, Thermofinnigan) coupled to a high performance liquid chromatograph (HP1100, Agilent).

· HPLC/PDA: High performance liquid chromatograph (HP1100, Agilent) with photodiode array detector

· MALDI-TOF-TOF: Matrix Assisted Laser Desorption Ionization Mass Spectrometer (4700, Applied Biosystems).

Funding

· Research Corporation “Elucidation of Echinacea’s Mechanism of Action: A Proteomic Approach” Nadja Cech, Principal Investigator (submitted, proposed dates 05/1/06-04/30/08)

· National Science Foundation, “Acquisition of a MALDI-TOF Mass Spectrometer for Research Involving Undergraduate Students” Nadja Cech, Principal Investigator, Gregory Raner, G. Brent Dawson, Jason Reddick, Ronald Morrison, Co-Principal Investigators (08/15/03-03/31/06)

· Research Corporation, “Identification of Immune Stimulating Liver Metabolites of Echinacea and Spilanthes” Nadja Cech, Principal Investigator (05/15/04-05/14/06)

· UNC Greensboro Regular Faculty Grant, “Drug Interactions with Echinacea and Spilanthes” Nadja Cech and Gregory Raner, Co-Principal Investigators (5/15/04-5/15/05)

· National Institutes of Health (Center for Complementary and Alternative Medicine) R15 Research Grant, “Synergy in Immunomodulation by Echinacea and Spilanthes” Nadja Cech, Principal Investigator, Cynthia Wenner Co-Investigator (08/03 - 05/06)

· North Carolina Institute of Nutrition Research Grant, “Drug Interactions with the Medicinal Herb Goldenseal: Relation to Cytochrome P450 Metabolism” Nadja Cech and Gregory Raner, Co-Principal Investigators (07/02-07/03)

· UNC Greensboro Summer Excellence Award, “Mass Spectrometry for the Comprehensive Characterization of Immune-Stimulating Medicinal Plants” Nadja Cech, Principal Investigator, (06/02 – 08/02)

· UNC Greensboro New Faculty Award, “Factors Influencing Active Constituent Levels in the Endangered Medicinal Plant Goldenseal (Hydrastis canadensis)” Nadja Cech, Principal Investigator, (01/02 - 05-02)

Publications coauthored by Nadja Cech

For reprint requests, please email nadja_cech@uncg.edu

1. Sasagawa, M.; Cech, N. B.; Gray, D. E.; Elmer, G. W.; Wenner, C. A. "Echinacea alkylamides inhibit interleukin-2 production by Jurkat human T cells" Int. Immunopharm. 2006, in press

2. Cech, N. B.; Enke, C. G. In Encyclopedia of Mass Spectrometry; "Electrospray ionization mass spectrometry: How and when it works"; Gross, M. L., Caprioli, R., Eds.; Pergamon: New York, 2006; Vol. 8, in press.

3. Cech, N. B.; Eleazer, M. S.; Shoffner, L. T.; Davis, A. C.; Crosswhite, M. R.; Mortenson, A. M. "High performance liquid chromatography/electrospray ionization mass spectrometry for simultaneous analysis of alkylamides and caffeic acid derivatives from Echinacea purpurea extracts" J. Chromatogr. A. 2006, 1103, 219-228.

4. Henriksen, T. R.; Juhler, R. K.; Svensmark, B.; Cech, N. B. "The relative influences of acidity and polarity on responsiveness of small organic molecules to analysis with negative ion electrospray ionization mass spectrometry (ESI-MS)" J. Am. Soc. Mass Spectrom. 2005, 16, 446-455.

5. Cech, N. B.; Enke, C. G. "Practical implications of some recent studies in electrospray ionization fundamentals" Rev. Mass Spectrom. 2002, 20, 362-287.

6. Cech, N. B.; Krone, J. R.; Enke, C. G. "Predicting electrospray response from chromatographic retention time" Anal. Chem. 2001, 73, 208-213.

7. Cech, N. B.; Enke, C. G. "The effect of affinity for charged droplet surfaces on the fraction of analyte charged in the electrospray process" Anal. Chem. 2001, 73, 4632-4639.

8. Cech, N. B.; Krone, J. R.; Enke, C. G. "Electrospray ionization detection of an inherently nonresponsive epoxide by peptide binding" Rapid Commun. Mass Spectrom. 2001, 15, 1040-1044.

9. Amad, M. H.; Cech, N. B.; Jackson, G. S.; Enke, C. G. "Importance of gas phase proton affinities in determining the electrospray ionization response for analytes and solvents" J. Mass Spectrom. 2000, 35, 784-789.

10. Cech, N. B.; Enke, C. G. "Relating Electrospray Ionization Response to Non-polar Character of Small Peptides" Anal. Chem. 2000, 72, 2717-2723.