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.
Research has continued utilizing available resources to produce the dehydropyrrolizidine (DHPAs) on a larger scale. Bulk comfrey and selected Amsinckia plant material has been processed to supply pure lycopsomine and intermedine that can now be used to support validation of analytical methods. Efforts are being directed to supply the acetylated derivatives either from plant material or conversion of lycopsomine and/or intermedine. A plant source (Echium vulgare) of echimidine was located in Montana and a bulk collection of this material was made. This material is now be extracted in bulk and being processed to provide significant quantities of this alkaloid.
As supplementary projects, purified alkaloids have been supplied in support of an assessment of relative toxicities of DHPAs and some N-oxides. Projects include both in vitro cytotoxicity and in vivo carcinogencity assays.