Lecturer University of Texas Rio Grande valley Edinburg, Texas
Body of Abstract: Tall fescue (Lolium arundinaceum, Schreb), is a major forage crop in the United States due to its resilience to extreme temperature conditions. Tall fescue has a symbiotic relationship with an endophytic fungus, Epichloë coenophiala, which enhances the stress tolerance of the host plants. The symbiotic fungus produces ‘ergot’ alkaloids (mainly ergovaline/ergovalinine) which cause several diseases in livestock. Hence, it is very important to have purified alkaloids to use as ‘standards’ during quantification of ergot alkaloids in tall fescue tissue samples and bioassays. Briefly, a stainless-steel tub was used as an extraction column where 23 kg of ground seed (passed through 1mm sieve size) was packed carefully. The ground seed was soaked in 80% ethanol for 5 days prior to collection, to increase the extraction efficiency. The extracted material was collected through an outlet, located at the bottom of the tub followed by freeze-drying to remove ethanol. A hexane/water cleanup of the extracted material was done to remove most of the lipid substances, followed by the extraction of the aqueous layer with chloroform. The alkaloid extraction with CHCl3 yielded a 270-fold increase in the concentration of total ergovaline and ergovalinine. After this, a silica gel column purification step was performed to remove other polar components from the extract, resulting in a 750-fold increase in the concentration of total ergovaline/ ergovalinine compared to their initial concentration in the seed. The purified fraction was further purified by a preparative reversed-phase HPLC using a linear binary gradient with 0.04% NH4OH in water and 100% acetonitrile resulting in a 5000-fold increase in the concentration of total ergovaline and ergovalinine compared to seed.