|FANNIN, F. NEIL - University Of Kentucky|
|JI, HUIHUA - University Of Kentucky|
|BUSH, LOWELL - University Of Kentucky|
Submitted to: International Symposium on Fungal Endophytes of Grasses
Publication Type: Abstract Only
Publication Acceptance Date: 4/19/2010
Publication Date: 6/28/2010
Citation: Fannin, F., Ji, H., Klotz, J.L., Bush, L.P. 2010. A Simple Tall Fescue Seed Extraction and Partial Purification of Ergovaline. International Symposium on Fungal Endophytes of Grasses. p 31.
Technical Abstract: There are several substances present in the tall fescue/endophyte association (Lolium arundinaceum /Neotyphodium coenophialum) that have biological activity. These include the pyrrolizidine and ergot alkaloids plus peramine. Of these compounds only the ergot alkaloids have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. As part of developing a repeatable in vivo model for studying effects of endophyte-infected tall fescue in mammalian systems, we developed a simple seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed were ground to pass a 2 mm sieve and were carefully packed into a 30 cm x 80 cm column. The bottom of the column contained approximately 4 cm of glass nuggets covered with an expanded metal screen and a Miracloth filter to keep ground seed above the glass nuggets. Approximately 25 kg of seed could be extracted in the column each time. Extraction solution was 80% ethanol and sufficient volume was added to fill void volume (~38 L) over ~ 6 hr. When solvent front migrated to bottom of the column, flow was stopped and seed steeped for at least 12 hr. Column was eluted with 80% ethanol at 2 L hr-1 for 25 hr. Ethanol was removed from the eluate in the dark by evaporation at room temperature. Resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration. This dried product was extracted with hexane/water (6:1, v/v) and the hexane discarded. The aqueous layer was extracted with chloroform, aqueous layer discarded and the chloroform was removed. Residue from the chloroform was freeze-dried and about 50% of ergovaline was recovered. Similar results were obtained with ergovalinine. The partially purified ergovaline had biological activities in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.