Location: Poisonous Plant Research
2012 Annual Report
However, without ample quantities of the standards, any application of the resultant assay is compromised. Therefore, the research has been directed to utilizing available resources to produce the DHPAs on a larger scale. This has resulted in the evolution of a new stage in the “clean-up” of comfrey extracts that involves a partitioning with dilute acid and n-butanol, the latter removing the large amounts of complicating co-extractives from the DHPAs and their N-oxides.
The crude comfrey root DHPA extract consists of four groups of diastereoisomers. Separation of the components within each group has been the major challenge. Fortuitously, other DHPA-related problems have presented the opportunity to experiment with separation approaches without risking the stocks of comfrey root crude extracts while simultaneously allowing a furthering of DHPA-related knowledge in the literature. These include: a collaborative study of Cryptantha crassipes that determined high levels of lycopsamine-N-oxide in the endangered plant from Texas; a study of Crotalaria juncea (sunn hemp) that unequivocally identified a suite of new DHPAs and confirmed general low levels of DHPAs in the seed commercially available for agricultural use; an investigation of a recent cattle poisoning incident in Arizona that potentially involved Amsinckia menziesii (intermedia). Contrary to all literature reference, analysis showed this Amsinckia chemotype to be a very valuable source of lycopsamine-N-oxide. This is currently being extracted to provide the pure lycopsamine for the comfrey study; two Cryptantha sp. collected in the vicinity of the Arizona poisoning incident were analyzed using the HPLC-esi(+)MS approach. One species produces echimidine and related DHPAs, including a potentially toxic DHPA that was unequivocally identified for the first time, and may provide a source for the standards required for the major comfrey species Symphytum uplandicum. The other Cryptantha produces a previously undescribed, and potentially toxic, alkaloid as the major DHPA. Isolation of this DHPA allowed a successful testing of new silica columns that recently became available for the Biotage flash chromatography system and that may assist in the comfrey project. The structure has been unambiguously elucidated using MS and NMR data.