Location: Poisonous Plant Research2011 Annual Report
1a. Objectives (from AD-416)
Research will be conducted to demonstrate a generically-applicable method for the quantitative analysis of pyrrolizidine alkaloids and their N-oxides in plant-derived products. Methods for the extraction, concentration, identification and quantitative analysis of the major Symphytum app. pyrrolizidine alkaloids and their N-oxides will be developed and optimized. This project is a continuation and extension of the previous project funded from March 2009 to March 2011.
1b. Approach (from AD-416)
The entire analytical process will be validated for separate Symphytum sp. based matrices according to the AOAC International Guidelines for Single Laboratory Validation of Chemical Methods for Dietary Sypplements and Botanicals. Standard reference materials will be prepared by isolation and purification of the major pyrrolizidine alkaloids from Symphytum sp. Authentication of standards will include description of characteristics such as physico-chemical properties (e.g. melting point, optical rotation and chromatographic profile), the molecular formula as determined using high resolution mass spectrometry, and a complete structure mapping using nuclear magnetic resonance spectroscopy.
3. Progress Report
The supplies of comfrey root, both Symphytum officinale and S. uplandicum have been extracted and the alkaloid fraction reduced to produce dehydropyrrolizidine alkaloid (DHPA) free bases from the naturally-occurring N-oxides. These important comfrey species have different suites of DHPAs. Several and various ways to try and purify the alkaloids from each species have been investigated with varying degrees of success. The main problem and goal is the separation of closely related diastereoisomers which, especially echimidine the major DHPA in S. uplandicum, have a poor reputation for stability during chromatographic separations. The processing thus far has resulted in a fridge full of valuable (or at least precious) alkaloidal fractions in various stages of purification from each of the two comfrey species being investigated, all awaiting elucidation of the best way to progress the separations. To this end, a current collaborative project with USDA/NCRS researchers in Hawaii is investigating extracts of Crotalaria juncea (Sunn Hemp). While containing different DHPAs to the DHPA monoesters and open ring diesters of comfrey, it is providing some good insights into the use of the Biotage Isolera chromatographic equipment for effective separation. In addition, some previously undescribed DHPA monoester diastereoisomers have been detected in C juncea and tentatively identified using mass spectrometry. The unambiguous structure elucidation of one of these diastereoisomers, isolated in pure enough form to obtain NMR data, has provided a very useful exercise in the collaborative use of the Utah State University NMR facilities in preparation for the unambiguous characterization of comfrey alkaloids. The HPLC-esiMS analytical method planned for validation with the comfrey alkaloids has also been applied to this collaborative study of C. juncea to ensure that a commercially available cultivar (Tropic Sunn), released by the USDA, is still low in the potentially toxic DHPAs.