Biological Activity, Analytical Detection, and Degradation Assessment of the S-epimers of Ergot Alkaloids

Ergotism has an extensive history dating back centuries. The fungus Claviceps purpurea produces ergot sclerotia that contain secondary metabolites called ergot alkaloids. Ergot alkaloids cause toxic effects after consumption of ergot contaminated food and feed. Ergot contaminated food and feed currently poses a health risk to humans and animals. There are six common ergot alkaloids produced by the fungus that exist in two configurations. The two configurations are the C-8-R-isomer (R-epimer) and the C-8-S-isomer (S-epimer), the latter of which has been studied to a lesser extent. The S-epimers have been considered inactive in terms of biological activity compared to the R-epimers. The R-epimers can elicit their toxic effects by binding to receptors in vascular smooth muscle cells. In this dissertation, the aim was to further understand the S-epimers of the six ergot alkaloids common to Claviceps purpurea through biological, analytical, and degradation assessments. An arterial tissue bath technique was used to assess the bioactivity of selected S-epimers. Following the exposure of arteries to the S-epimers, vascular constriction was observed. Exposure of increasing concentrations of the S-epimers to the arteries resulted in an increase in the contractile response. In a following study, arterial contraction due to the S-epimers over time was assessed and a sustained vascular contraction was observed. The sustained vascular contraction of the S-epimer was different than the corresponding R-epimer, which may be due to differences in vascular contraction mechanisms. This is the first time that the S-epimers of ergot alkaloids have demonstrated bioactivity, contrary to historical belief. To support the vasoconstriction observed in the previous studies, a novel approach using an in silico method to assess ergot structure activity relationships was used, specifically to observe the S-epimer-vascular receptor relationship. The S-epimers bound to vascular receptors with relatively high affinity and strong molecular interactions, which has not been documented previously. Since the S-epimers demonstrated bioactivity and can bind to vascular receptors, it was important to assess the concentrations of the S-epimers in natural ergot contaminated samples. A newly validated analytical method to detect and quantify R and S-epimer concentrations using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used. This new analytical method can be implemented in laboratories worldwide for the sensitive and accurate detection and quantification of ergot epimers. It was observed that the S-epimers of the six common ergot alkaloids constitute approximately 35% of the total ergot alkaloid concentration in the samples assessed. When the natural ergot contaminated samples were exposed to various temperatures over time, the R and S-epimer concentrations varied. Therefore, ergot contaminated samples should be analyzed prior to their use for food or feed to obtain the most accurate concentration results. The high concentration of the S-epimers and the lack of concentration stability warranted an investigation into the degradation of the S-epimers associated with their potential bioactivity which can lead to toxic effects. Some studies assessing degradation methods of ergot alkaloids only quantify or focus on the R-epimers and not the S-epimers. Through using degradation methods of ammonia and ultra-violet (UV) light, based on practicality, we observed that the R and S-epimer concentrations behave differently, following the degradation methods. Ammonia significantly reduced the concentration of total ergot alkaloids, while demonstrated varied effects on the concentrations of the R and S-epimers. The use of ammonia for the degradation of ergot alkaloids may be a practical means for livestock to consume ergot contaminated feed without any adverse effects. The use of UV light did not significantly alter the concentrations of the total ergot alkaloids; however, it decreased the concentration of the S-epimer, ergotaminine. Since the effects of the degradation methods on the R and S-epimer concentrations may vary, it is important to assess both configurations following degradation assessments. Overall, this research has furthered our understanding of the S-epimers of ergot alkaloids. Continuous research on the S-epimers of ergot alkaloids is needed to further understand the S-epimers in terms of their bioactivity, stability, and degradation. It was important to investigate the S-epimers since they were historically deemed as non-bioactive and have not been thoroughly researched, compared to the R-epimers. The results of this dissertation have demonstrated, for the first time, the bioactivity of the S-epimers and the use of a novel in silico method to assess how the S-epimers may contribute to the adverse effects associated with ergot alkaloids. In addition, high concentrations of S-epimers were observed within ergot contaminated samples and the use of ammonia as a method to degrade ergot epimers may be practical within the agriculture industry. Guidelines for the concentrations of ergot alkaloids in food and feed have been set worldwide; however, some guidelines only include the R-epimers and not the S-epimers. The results of this dissertation encourage the inclusion of the S-epimers into guidelines globally for both food and feed for the protection of human and animal health. The observation that the S-epimers are bioactive, have high concentrations in contaminated matrices, and may contribute to the toxic effects following ergot alkaloid consumption, will ultimately improve food and feed safety guidelines for both human and livestock populations.

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