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%).
Grancher
Denis
D
UMR 1233 INRA-ENVL, Mycotoxins and Comparative Toxicology of Xenobiotics, National School of Veterinary Medicine of Lyon, I Avenue Bourgelat, BP 83, 69280 Marcy l'Etoile, France. d.grancher@vet-lyon.fr
Jaussaud
Philippe
P
Durix
Andrée
A
Berthod
Alain
A
Fenet
Bernard
B
Moulard
Yves
Y
Bonnaire
Yves
Y
Bony
Sylvie
S
D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003377:Countercurrent Distribution / Q000379:methods*; D026121:Indole Alkaloids; D008129:Lolium / Q000737:chemistry* / Q000196:embryology; D009682:Magnetic Resonance Spectroscopy; D009183:Mycotoxins / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry*
D026121:Indole Alkaloids; D009183:Mycotoxins; C033673:lolitrem B