Countercurrent chromatographic isolation of lolitrem B from endophyte-infected ryegrass (Lolium perenne L.) seed

This paper describes a new method of purification of the Lolitrem B, a tremorgenic mycotoxin produced in planta by the endophytic fungus Neotyphodium lolii. The method is based on the large-scale isolation of the toxin by countercurrent chromatography (CCC). The lolitrem B content in endophyted ryegrass seed, 11 microg/g or 11 ppm, is extracted by stirring finely ground seeds with ethanol for 3 h at room temperature. The concentrated crude extract contains about 0.6 mg/g or 600 ppm of lolitrem B. It is then submitted to CCC purification with a biphasic four-solvent liquid system. A 160-fold enrichment was obtained in one step producing a raffinate containing 10% or 100 mg/g of the toxin. Further purifications were then performed by thin layer and low pressure liquid chromatography. Twenty-eight micrograms of lolitrem B with a 96% purity grade were obtained from 8 kg of seeds (yield 32%).