|HUIHUA, JI - University Of Kentucky|
|FANNIN, NEIL - University Of Kentucky|
|BUSH, LOWELL - University Of Kentucky|
Submitted to: Frontiers in Chemistry: Chemical Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/21/2014
Publication Date: 12/11/2014
Citation: Huihua, J., Fannin, N., Klotz, J.L., Bush, L.P. 2014. Tall fescue seed extraction and partial purification of ergot alkaloids. Frontiers in Chemistry: Chemical Biology. 2:110.
Interpretive Summary: The symbiotic relationship of tall fescue (Schedonorus arundinaceus) and the fungal endophyte (Epichloë coenophiala) results in the production of numerous secondary metabolites. The biologically active substances include peramine, pyrrolizidine alkaloids and ergot alkaloids. Of these compounds, only the ergot alkaloids have significant mammalian toxicity and the predominant produced ergot alkaloids are ergovaline and ergovalinine. These two ergopeptine alkaloids are isomers and in equilibrium depending upon the environment in which they are contained. Ergovaline is the more biologically active of the two isomers and neither are easily chemically synthesized. Chemical standards for analytical assays or for bioassays are very difficult to obtain. Therefore, our objective was to isolate and partially purify ergovaline/ergovalinine from tall fescue seed infected with the endophyte. Solvent, seed particle size, and time used for extraction had significant impact of efficiency of extraction. Overall this is a protocol for extraction of large amount of high ergovaline plant tissue that yields dried extracts with enhanced levels of ergovaline that are biologically active. Activity is equal to ergovaline alone in selected bioassays. Additional purification can be achieved with HPLC separation of ergovaline and ergovaline, plus lysergic acid and isolysergic acid. This further purification of ergovaline will be useful in specific cellular bioassays and for analytical purposes.
Technical Abstract: Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W×L×D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. Packing the column was done to insure that the solvent would migrate uniformly to the bottom of the column. This was done by adding about 22 kg at a time, leveling and lightly packing. The extraction was done with 80% ethanol. To fill the void volume, approximately 160 L were added over an 18 to 20 h period. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature. Resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v) and the hexane fraction was discarded. The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed. Residue from the chloroform and about 65% of ergovaline was recovered for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.