Kentucky Tobacco Research and Development Center, University of Kentucky Lexington, KY, USA.
Department of Plant and Soil Sciences, University of Kentucky Lexington, KY, USA.
Front Chem. 2014 Dec 11;2:110. doi: 10.3389/fchem.2014.00110. eCollection 2014.
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. The extraction was done with 80% ethanol. 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 and the resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v). The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue 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.
高羊茅/内生真菌（羊茅属/内生赤霉）中的许多物质都具有生物活性。在这些化合物中，只有麦角生物碱被认为对哺乳动物具有显著的毒性，而主要的麦角生物碱是麦角新碱和麦角隐亭。由于合成的麦角新碱难以获得，我们开发了一种从麦角新碱/麦角隐亭中提取和部分纯化的方法，该方法提供了一种具有生物活性的产物。高羊茅种子被研磨并装入几个不同大小的柱中进行液体提取。较小的颗粒尺寸和增加的提取时间提高了提取效率。我们最大的柱子是一个 114×52×61cm（W×L×D）的不锈钢桶。这个桶可以提取大约 150 公斤的种子。提取溶剂是 80%的乙醇。当溶剂前沿迁移到柱子底部时，停止流动，让种子浸泡至少 48 小时。从这一步开始到纯化过程结束，都要避免光照进入溶剂。洗脱后，在室温下蒸发去除洗脱液中的乙醇，得到的糖浆用冷冻干燥。通过 18 倍的浓度增加，大约 80%的生物碱得到回收。通过用正己烷/水（6:1，v/v）提取，对干燥产物进行初步纯化。将水相用氯仿萃取，丢弃水层，然后用氯仿萃取，得到 20 倍的麦角新碱。从氯仿残留物中回收了约 65%的麦角新碱，总回收率为 50%。所得部分纯化的麦角新碱在体内和体外牛生物测定中具有与合成麦角新碱相近的生物活性。
